Cell preparation composition, system and method for single cell transcriptomics

Abstract

Disclosed herein are aspects of a composition, typically comprising a functionalized magnetic particle with an outer surface comprising a ligand on the outer surface, wherein the ligand is bound to a permeabilized cell comprising a nucleic acid for generating a nucleic acid library. Also disclosed are a method of tagging a nucleic acid within at least one cell, a method of generating a nucleic acid library, systems configured for processing disclosed compositions and implementing disclosed methods, and kits comprising a composition or compositions for use with disclosed method aspects.

Description

[0001] CELL PREPARATION COMPOSITION, SYSTEM AND METHOD FOR SINGLE CELL

[0002] TRANSCRIPTOMICS

[0003] CROSS REFERENCE TO RELATED APPLICATION

[0004] 5 This application claims the benefit of the earlier filing date of U.S. provisional patent application No. 63 / 533,464, filed August 18, 2023, which is incorporated herein by reference in its entirety.

[0005] FIELD

[0006] The present specification concerns a composition, system, method, and kit for

[0007] 10 preparing a cell, such as for single cell transcriptomics.

[0008] BACKGROUND

[0009] Single-cell transcriptomics currently requires oil drop-based isolation technologies or laborious centrifugation. Moreover, current technological requirements of centrifugation and oil-droplet isolation of single cells do not allow for high- throughput automation methodologies. For example, the SPLiT-seq workflow requires 10 hours or more to complete including laborious centrifugation. SPLiT-seq is a method for single cell or nuclei transcriptome analysis that uses crosslinking, template switching, incorporating barcodes via ligation, and Nextera fragmentation to prepare a 0 library for nucleic acid sequencing. Due to the multiple centrifugation wash steps required, the SPLiT-seq workflow cannot be automated. Eliminating the equipment required for the centrifugation or oil-drop based technologies would allow for high- throughput automation. Therefore, there is a need to eliminate cost-prohibitive, oil drop-based isolation technologies, eliminate laborious centrifugation, and to reduce the workflow time required in single-cell transcriptomics.

[0010] Additionally, current on-bead assays do not address single-cell transcriptomics. For example, CUT&RUN epigenetic assay utilizes target-specific primary antibodies and pAG-MNase to isolate protein DNA complexes on native chromatin for library development. Moreover, CUT&TAG investigates interactions between proteins and 0 DNA to identify binding sites on the DNA for a protein of interest, which is transposase-based epigenetic library development. CUTAC, another epigenetic assay, is a bead-based technique for identifying open chromatin with Tn5 Transposase. Thus, there is a need for on-bead methodologies that allow for single-cell transcriptomics because current technologies focus solely on the interrogation of epigenetic marks within the DNA and genome.

[0011] SUMMARY

[0012] Disclosed aspects of the present disclosure advantageously provide superior aspects of a workflow for developing nucleic acid libraries for single-cell transcriptomics by using functionalized magnetic particles bound to at least one permeabilized cell via a ligand. Disclosed aspects also additionally reduce time and expense associated with developing such nucleic acid libraries.

[0013] In some aspects, provided herein are methods for tagging a nucleic acid within a cell, comprising:

[0014] (a) contacting a permeabilized cell comprising at least one cellular nucleic acid and a functionalized magnetic particle comprising at least one ligand, to produce a mixture comprising the permeabilized cell associated with the at least one ligand of the functionalized magnetic particle;

[0015] (b) contacting the at least one cellular nucleic acid with a first oligonucleotide, at least one nucleotide, a first primer, and a first polymerase under conditions to produce a first tagged nucleic acid strand in the permeabilized cell; and

[0016] (c) applying a magnetic field to the mixture, thereby separating the permeabilized cell associated with the ligand of the functionalized magnetic particle and the first tagged nucleic acid strand from other components of the mixture; wherein steps (a) and (b) can occur in either order.

[0017] In some examples, the ligand of the functionalized magnetic particle is a carbohydrate or a lectin. In one example, the ligand is Concanavalin A.

[0018] In some aspects, the methods further include contacting the functionalized magnetic particle comprising at least one ligand with an activation buffer to increase affinity of the permeabilized cell to the functionalized magnetic particle. In additional aspects, the permeabilized cell is prepared by contacting the cell with a permeabilizing agent to form the permeabilized cell. In some examples, the permeabilized cell is contacted with a fixation agent or is fixed prior to permeabilization and / or prior to contacting the permeabilized cell with the functionalized magnetic particle comprising the at least one ligand. The methods may further include providing a quenching solution to the fixation agent.

[0019] In some aspects, the at least one oligonucleotide is at least one oligonucleoti detethered nucleotide. The nucleotide may include dideoxyadenosine triphosphate, dideoxyguanosine triphosphate, dideoxythymidine triphosphate, dideoxyuridine triphosphate, dideoxycytidine triphosphate, or any combination thereof. In certain aspects, the oligonucleotide-tethered nucleotide comprises Formula 1, or a salt thereof

[0020] Formula 1, wherein NB is a nucleobase; each of X and Q are independently selected from, H, OH, N3, halo, alkyl, alkoxy, alkyl, alkenyl, alkynyl, acyl, cyano, amino, ester, and amido;

[0021] Z and Y are independently a bond, amino, amido, alkyl, alkenyl, alkynyl, thioether, sulfonyl, sulfonamido, ether, ketone, carbonyl, anhydride, ester, imido, urea, urethane, and combinations thereof; and

[0022] CXN is selected from alkylene, alkenylene, alkynylene, ketone, carbonate, ester, ether, anhydride, amido, amino, aminoalkylene, imino, imido, diazo, carbamate ester, phosphodiester, sulfide, disulfide, sulfonyl, sulfonamido, and a heterocyclic group containing from one to four N, O, S atom or a combination thereof.

[0023] In additional aspects, the methods further include contacting the first tagged nucleic acid strand with a second primer, wherein the second primer is partially complementary to the oligonucleotide-tethered nucleotide after producing the first tagged nucleic acid strand, to form an annealed second primer; contacting the first tagged nucleic acid strand and the annealed second primer with a second polymerase and at least one nucleotide not tethered to an oligonucleotide to extend the oligonucleotide-tethered nucleotide sequence, using the second primer as a template to form a second tagged nucleic acid strand, wherein the second tagged nucleic acid strand is present in a mixture comprising the permeabilized cell associated with the ligand of the functionalized magnetic particle; and placing the mixture comprising the permeabilized cell associated with the ligand of the functionalized magnetic particle and the second tagged nucleic acid strand in a magnetic field, thereby separating the permeabilized cell associated with the ligand of the functionalized magnetic particle and the second tagged nucleic acid strand from other components of the mixture.

[0024] In other aspects, the methods further include contacting the first tagged nucleic acid strand with a double-stranded oligonucleotide, wherein one of the nucleotides is partially complementary to the oligonucleotide-tethered nucleotide after producing the first tagged nucleic acid strand, to form an annealed second primer; ligating the non- complementary strand of the double-stranded oligonucleotide to the oligonucleotide- tethered nucleotide, thereby producing a second tagged nucleic acid strand, wherein the second tagged nucleic acid strand is present in a mixture comprising the permeabilized cell associated with the ligand of the functionalized magnetic particle; and placing the mixture comprising the permeabilized cell associated with the ligand of the functionalized magnetic particle and the second tagged nucleic acid strand in a magnetic field, thereby separating the permeabilized cell associated with the ligand of the functionalized magnetic particle and the second tagged nucleic acid strand from other components of the mixture.

[0025] In some aspects, the first primer, the second primer, and / or the tethered oligonucleotide comprise a random sequence, a target-specific sequence, or both. In certain examples, the first primer, the second primer, and / or the tethered oligonucleotide include a universal handle, a universal sequence, a unique molecular identifier, an adapter sequence, a promoter sequence, a barcode sequence, an index sequence, or any combination thereof. Also provided are methods for preparing a nucleic acid library, the method comprising:

[0026] 16. A method for preparing a nucleic acid library, comprising: preparing a sample comprising a plurality of cells, wherein at least one cell of the plurality of cells comprises a cellular nucleic acid, and wherein the at least one cell is fixed and permeabilized; barcoding the cellular nucleic acid of the at least one fixed and permeabilized cell in the mixture; contacting the sample with a functionalized magnetic particle comprising at least one ligand, producing a mixture wherein the at least one fixed and permeabilized cell is bound to the at least one ligand; applying a magnetic field to the sample, thereby separating the mixture from the other components of the sample; lysing the plurality of cells in the separated mixture; and preparing a nucleic acid library from the lysed cells by providing at least one amplification primer to form an amplified nucleic acid.

[0027] In some aspects, barcoding the cellular nucleic acid includes: splitting the mixture into a plurality of first containers; preparing nucleic acids that are complementary to the cellular nucleic acid; annealing a first primer to form an annealed first primer which is at least partially complementary to the at least one complementary nucleic acid, the first primer comprising a first universal handle sequence and a first barcode, the first barcode being common to the container, but different from the first barcodes present in the first primers in other containers; and contacting the at least one complementary nucleic acid with a polymerase, at least one nucleotide, and at least one oligonucleotide-tethered dideoxynucleotide, the oligonucleotide of the oligonucleotide-tethered dideoxynucleotide comprising a second universal handle sequence. In additional aspects, the barcoding further includes: forming at least one nucleic acid strand comprising the oligonucleotide-tethered dideoxynucleotide at their 3’ end; pooling the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the least one nucleic acid strands comprising the oligonucleotide-tethered dideoxynucleotide at their 3’ end from the plurality of first containers to provide a first pool; washing the first pool; splitting the first pool into a plurality of second containers; annealing a second primer with the tethered oligonucleotide to form an annealed second primer, wherein the second primer is at least partially complementary to the tethered oligonucleotide, and allowing the polymerase to extend from a 3’ hydroxyl of the annealed second primer to the tethered oligonucleotides to form an extended annealed second primer; pooling the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the extended annealed second primer to provide a second pool; washing the second pool; and splitting the second pool into a plurality of third containers.

[0028] In additional aspects, the barcoding further includes: forming a first extension product comprising the oligonucleotide-tethered dideoxynucleotide at the 3’ end; pooling the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the formed first extension products comprising the first extension product to form a third pool; washing the third pool; splitting the third pool into a plurality of fourth containers; contacting a splint oligonucleotide with the tethered oligonucleotide of the first extension product, wherein the splint oligonucleotide is partially complementary to the tethered oligonucleotide of the first extension product; contacting the first extension product with a nucleic acid polymerase and one or more nucleotides to allow the polymerase extend across the annealed splint from the 3’ hydroxyl of the tethered oligonucleotide to produce a second extension product; pooling the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the second extension product to form a fourth pool; washing the fourth pool; and splitting fourth pool into a plurality of fifth containers.

[0029] In some aspects, barcoding the nucleic acid includes splitting the functionalized magnetic particles bound to at least one fixed and permeabilized cell into a plurality of first containers; contacting the nucleic acid with an enzyme to generate an at least one complementary nucleic acid; providing a first primer to form an annealed first primer which is at least partially complementary to the at least one complementary nucleic acid, the first primer comprising a first universal handle sequence and a first barcode, the first barcode being common to the container, but different from the first barcodes present in the first primers in other containers; and contacting the at least one complementary nucleic acid with a polymerase, at least one nucleotide not tethered to an oligonucleotide, and at least one oligonucleotide-tethered dideoxynucleotide, the oligonucleotide of the oligonucleotide-tethered dideoxynucleotide comprising a second universal handle sequence.

[0030] In some aspects, the barcoding further includes forming at least one nucleic acid strand comprising the oligonucleotide-tethered dideoxynucleotide at their 3’ end; pooling the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the least one nucleic acid strands comprising the oligonucleotide-tethered dideoxynucleotide at their 3’ end; washing the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the least one nucleic acid strands comprising the oligonucleotide-tethered dideoxynucleotide at their 3’ end; splitting the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the least one nucleic acid strands comprising the oligonucleotide-tethered dideoxynucleotide at their 3’ end into a plurality of second containers; contacting a second primer with the tethered oligonucleotide to form an annealed second primer, wherein the second primer is at least partially complementary to the tethered oligonucleotide, and allowing the polymerase to extend from a 3’ hydroxyl of the annealed second primer to the tethered oligonucleotides to form an extended annealed second primer; pooling the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the extended annealed second primer; washing the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the extended annealed second primer; and splitting the functionalized magnetic particles bound to the fixed permeabilized cell comprising the extended annealed second primer into a plurality of third containers.

[0031] In other aspects, the barcoding further includes forming a first extension product comprising the oligonucleotide-tethered dideoxynucleotide at the 3’ end; pooling the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the formed first extension products comprising the first extension product; washing the functionalized magnetic particles bound to the fixed and permeabilized cell comprising first extension products comprising the first extension product; splitting the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the first extension product into a plurality of second containers; contacting a splint oligonucleotide with the tethered oligonucleotide of the first extension product, wherein the splint oligonucleotide is partially complementary to the tethered oligonucleotide of the first extension product; contacting the first extension product with a nucleic acid polymerase and one or more nucleotides to allow the polymerase extend across the annealed splint from the 3’ hydroxyl of the tethered oligonucleotide to produce a second extension product; pooling the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the second extension product; washing the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the second extension product; and splitting the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the extension product into a plurality of third containers.

[0032] In other examples, the barcoding further includes forming a first extension product comprising the oligonucleotide-tethered dideoxynucleotide at the 3’ end; pooling the functionalized magnetic particles bound to a permeabilized cell comprising the formed first extension products comprising the first extension product; washing the functionalized magnetic particles bound to a permeabilized cell comprising first extension products comprising the first extension product; splitting the functionalized magnetic particles bound to a permeabilized cell comprising the first extension product into a plurality of second containers; providing a pre-annealed oligonucleotide comprising a second barcode sequence which is at least partially complementary to the tethered oligonucleotide of the first extension products; contacting the first extension products with the pre-annealed oligonucleotide and a ligase to form a ligation product comprising the oligonucleotide-tethered dideoxynucleotide at the 3’ end and a second barcode; pooling the functionalized magnetic particles bound to a permeabilized cell comprising the ligation product; washing the functionalized magnetic particles bound to a permeabilized cell comprising the ligation product; and splitting the functionalized magnetic particles bound to a permeabilized cell comprising the ligation product into a plurality of third containers.

[0033] In some aspects, providing at least one amplification primer includes the at least one amplification primer hybridizing and extending from a first universal handle and a third universal handle. In some examples, the amplification primers include a third barcode, a fourth barcode, a first adaptor sequence, a second adapter sequence, or any combination thereof. In some examples, the methods further include generating amplification products, the combination of the first barcode sequence, the second barcode sequence, the third barcode sequence of the amplification products comprising unique sequences to the amplification products originating from a single cell or nucleus. Also provided herein are compositions including a functionalized magnetic particle comprising at least one ligand; at least one cell bound to the at least one ligand, wherein the cell comprises at least one cellular nucleic acid; and at least one nucleic acid complementary to the at least one cellular nucleic acid, wherein the complementary nucleic acid comprises a first barcode and an oligonucleotide tethered nucleotide. In some examples, the complementary nucleic acid is a cDNA. In some aspects, the cell is a permeabilized cell. In other aspects, the at least one functionalized magnetic particle has a diameter of from greater than 0 gm to 100 gm or a diameter from 500 nm to 1500 nm. In some examples, the at least one ligand comprises a lectin, for example, Concanavalin A.

[0034] In some aspects, the composition further includes at least one enzyme, for example a polymerase (such as a DNA polymerase or RNA polymerase), a ligase, or a reverse transcriptase. In some examples, the cellular nucleic acid is DNA, RNA, or both.

[0035] In additional examples, the at least one oligonucleotide tethered nucleotide includes from about 3 nucleotides to 100 nucleotides in length. In other examples, the at least one oligonucleotide-tethered nucleotide comprises Formula 1 (above), or a salt thereof. In some examples, the oligonucleotide-tethered nucleotide includes dideoxyadenosine triphosphate, dideoxyguanosine triphosphate, dideoxythymidine triphosphate, dideoxyuridine triphosphate, dideoxycytidine triphosphate, or any combination thereof.

[0036] Also provided are systems comprising: at least one extraction module; at least one container comprising a disclosed composition; a positioner to position the extraction module in the container to remove the composition from the container; and a controller having stored instructions for controlling the system.

[0037] In some aspects, the instructions comprise a method for preparing a single cell RNA sequencing library. In additional aspects, the container includes a columnar container, a tube, multi-well plate, or any combination thereof. In some examples, the extraction module includes at least one magnet.

[0038] Further provided herein are kits for preparing a nucleic acid library including a functionalized magnetic particle comprising at least one ligand; and at least one reaction component for incorporating an oligonucleotide into a nucleic acid of a cell in a sample via a synthesis reaction, wherein the oligonucleotide is an oligonucleotide-tethered nucleotide having Formula 1 (above), or a salt thereof. In some aspects, the ligand is a lectin, for example, Concanavalin A. In some aspects, the kit further includes a container comprising the at least one functionalized magnetic particle, at least one oligonucleotide, and the at least one reaction component. In other aspects, the kit further includes one or more containers including one or more of a binding buffer for activating the at least one functionalized magnetic particle, a buffering agent, a washing buffer, and a lysis buffer. In some aspects, the kit is configured for use with a system disclosed herein.

[0039] The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1 is a schematic drawing illustrating one disclosed aspect for preparing a nucleic acid library comprising using a functionalized magnetic particle bound to a cell and at least one reaction component.

[0042] FIG. 2 is a schematic drawing illustrating one disclosed aspect for preparing a nucleic acid library comprising using a functionalized magnetic particle bound to a permeabilized cell comprising at least one reaction component.

[0043] FIG. 3 is a schematic view of one disclosed aspect for preparing a nucleic acid library using a system according to the present disclosure comprising an extraction module, a container, and at least one functionalized magnetic particle bound to a cell.

[0044] FIG. 4 is a schematic perspective view of one disclosed aspect for preparing a nucleic acid library using a system according to the present disclosure comprising a magnet configured to a plural well-plate.

[0045] FIG. 5 is a schematic perspective view of one disclosed aspect for preparing a nucleic acid library using a system according to the present disclosure further comprising a rack.

[0046] FIG. 6 is a schematic perspective view of one disclosed aspect for preparing a nucleic acid library using a system according to the present disclosure comprising a rack configured for use with a magnet. FIG. 7 is a schematic perspective view of one disclosed aspect of a system according to the present disclosure comprising a storage container, a container comprising at least one or more troughs, plural 96-well plates, and an extraction module.

[0047] FIG. 8 is an enlarged schematic perspective view of one disclosed aspect of a system comprising a multi-well container, the wells of the container comprising a tapered end that can be configured for use with an extraction module to provide superior retention and extraction of functionalized magnetic particles bound to cells.

[0048] FIG. 9 is a flow chart illustrating one disclosed aspect of a split-pool method for using a functionalized magnetic particle coated with a ligand for preparing a nucleic acid library.

[0049] FIG. 10 illustrates one disclosed aspect illustrating an example workflow with ligation for preparing a nucleic acid library using a functionalized magnetic particle comprising a Concanavalin A ligand bound to a permeabilized cell comprising an oligonucleotide-tethered dideoxynucleotide, wherein contacting the mRNA with reverse transcriptase to generate cDNA (Step 1), the split-pool and ligation with the extension of barcode (Step 2), and cell lysis (Step 3), occur within a permeabilized cell, and thus can be used for bulk cell processing and multiplexing.

[0050] FIG. 11 is a graph of sample intensity versus base pair size for HEK / 3T3 cells.

[0051] FIG. 12 a graph of sample intensity versus base pair size for PBMC cells.

[0052] FIG. 13 illustrates one disclosed aspect illustrating an example workflow without ligation for preparing a nucleic acid library using a functionalized magnetic particle comprising a Concanavalin A ligand bound to a permeabilized cell comprising an oligonucleotide-tethered dideoxynucleotide, wherein contacting the mRNA with reverse transcriptase to generate cDNA (Step 1), split-pool and extension of barcode (Step 2), and split-pool and cell lysis (Step 3), occur within a permeabilized cell, which can be used for bulk cell processing and multiplexing.

[0053] FIG. 14 is a digital image made using Countess software depicting functionalized magnetic particles (black dots) comprising a Concanavalin A ligand bound to cells (blue dots), illustrating the affinity of functionalized magnetic particles comprising a Concanavalin A ligand to bind to a cell.

[0054] FIGS. 15-20 are images made using an Evos imaging system depicting cells bound to functionalized magnetic particles via the ligand Concanavalin A, which illustrate the affinity of functionalized magnetic particles with a Concanavalin A ligand to bind to a cell.

[0055] FIGS. 21 -26 are images made using an Cytpix imaging system depicting cells bound to functionalized magnetic particles via the ligand Concanavalin A, which illustrate the affinity of functionalized magnetic particles with a Concanavalin A ligand to bind to a cell.

[0056] FIG. 27 is a graph illustrating cell retention data (%) of one disclosed aspect comprising an “Eppendorf ConA” wash with functionalized Concanavalin A coated magnetic particles (without centrifugation), compared to cell retention for “Conical” cell wash with centrifugation and “Eppendorf’ cell wash also with centrifugation, with all three washes starting with 200,000 cells, where the “Conical” wash provided 50% to 75% cell retention; the “Eppendorf” wash provided 60% to 125% cell retention; and the “Eppendorf ConA” provided 70% to 120% cell retention, thereby illustrating the superior cell retention of cells washed via the functionalized Concanavalin A magnetic particles versus the cell washes with centrifugation (“Conical” and “Eppendorf’).

[0057] FIG. 28 is a graph depicting cell retention data (%) of one disclosed aspect comprising an automated high-throughput cell wash using a 96-well KingFisher™ Flex Purification System (Thermo Fisher) comprising cells bound to a Concanavalin A coated particle providing greater than 70% cell retention (“KingFisher”) versus the cell retention percentage of a centrifuge wash (“Centrifuge”) providing from 45% to 60% cell retention, illustrating superior cell retention by using the automated KingFisher™ Flex Purification System (Thermo Fisher) and functionalized Concanavalin A magnetic particles bound to cells.

[0058] FIG. 29 is a graph depicting cell retention data (%) of a first aspect comprising U937 cells (cell culture suspension cells, human monocytes) with a 75% to 115% cell retention; a second example aspect comprising PBMC cells (previously frozen human peripheral blood mononuclear cells in suspension) with a 100% to 120% retention; a third example aspect comprising Hek 293 cells (cell culture adherent cells, immortalized human embryonic kidney cells) with a 65% to 105% retention; and a fourth example aspect comprising 3T3 cells (cell culture adherent cells, mouse fibroblasts) with a 95% to 103% retention; illustrating the superior ability of functionalized Concanavalin A coated magnetic particles to bind to different cell types and to remain bound through a cell wash on a 96-well-plate automated workflow using the Kingfisher™ Flex Purification System. Values greater than 100% retention occur due to counting methods and do not indicate that cells were added to wells.

[0059] FIG. 30 is a graph depicting cell retention data (%) of a disclosed aspect for (1) a “Centrifuge” wash comprising 200,000 cells (starting) in a conical vial, (2) a “Control 100k” referring to 100,000 cells / well bound to a functionalized magnetic particle comprising a Concanavalin A ligand washed in a 96 well dish, and (3) “Control 10k” referring to 10,000 cells / well in a 96 well dish bound to a functionalized magnetic particle comprising a Concanavalin A ligand washed in a 96 well dish, illustrating superior retention of cells (greater than 70% retention) bound to functionalized magnetic particles comprising a Concanavalin A ligand washed at various cell concentrations.

[0060] DETAILED DESCRIPTION

[0061] I. Terms, Ranges, and Definitions

[0062] The following explanations of terms and abbreviations are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure.

[0063] As used herein, “comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements unless the context clearly indicates otherwise.

[0064] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.

[0065] The disclosure of numerical ranges should be understood as referring to each discrete point within the range, inclusive of endpoints, unless otherwise noted.

[0066] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term “about.” Accordingly, unless otherwise implicitly or explicitly indicated, or unless the context is properly understood by a person of ordinary skill in the art to have a more definitive construction, the numerical parameters set forth are approximations that may depend on the desired properties sought and / or limits of detection under standard test conditions / methods as known to those of ordinary skill in the art. When directly and explicitly distinguishing aspects from discussed prior art, the aspect numbers are not approximates unless the word “about” is recited.

[0067] Any of the groups referred to herein may be optionally substituted by at least one, possibly two or more, substituents as defined herein, unless the context indicates otherwise, or a particular structural formula precludes substitution.

[0068] “Amino” refers to -NR'R2, where R1and R2independently are selected from hydrogen and Ci-Cs alkyl groups.

[0069] “Alkyl” or “alkylene” refer to a radical derived from a saturated, linear or branched hydrocarbon chain, comprising for example, from 1 to 12 carbon atoms, or 1 to 6 carbon atoms, 1 to 4 carbon atoms or 2 to 3 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2- pentyl, 3-pentyl, 2-methyl butyl, 3-methyl butyl, and n-hexyl and any isomers, methylene, ethylene, propylene, isopropylene, n-butylene, pentylene, and the like.

[0070] “Alkenyl” and “alkenylene” refer to a radical group derived from a straight or branched hydrocarbon chain comprising 2 to 10 carbon atoms and containing at least one carbon-carbon double bond. Examples include ethenyl (vinyl), 1 -propenyl, 2- propenyl (allyl), isopropenyl, butenyl, buta- 1,4-dienyl, pentenyl, hexenyl, ethenylene), propenylene, butenylene, hexenylene, and the like.

[0071] “Alkynyl” or “alkynylene” refer to a divalent group derived from a straight or branched hydrocarbon chain comprising 2 to 10 carbon atoms containing at least one carbon-carbon triple bond.

[0072] "Alkoxy” refers to -OR wherein R is a Ci-8 alkyl group. Examples include - OCi-salkyl, such as — OMe (methoxy), — OEt (ethoxy), — O(nPr) (n-propoxy), — O(iPr) (isopropoxy), — O(nBu) (n-butoxy), — O(iBu) (isobutoxy), — O(tBu) (tert-butoxy), and the like.

[0073] “Azide” or “azido” refers to -N3, or — N=N =N’, or — N’— N =N.

[0074] “Barcode” refers to a first known nucleic acid sequence that is associated with a second nucleic acid sequence and may allow the identification or tracking of the second nucleic acid sequence, and / or may allow some feature of the second nucleic acid with which the barcode is associated to be identified. In some aspects, the feature of the nucleic acid to be identified is the sample or source from which the nucleic acid is derived. By way of example only, some aspects described herein describe the addition of multiple barcodes, which can be 2, 3, 4, 5, 6, or more barcodes, to nucleic acids of interest in a single cell present in a population of cells. The unique combination of barcodes added to the nucleic acids of each individual cell can advantageously enable the identification of the cell from which the tagged nucleic acid of interest was derived. In some aspects, suitable barcodes are from 3 to at least 15 nucleotides in length, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleotides in length. In some aspects, a barcode or barcodes associated with some nucleic acids are of a different length than barcodes associated with other nucleic acids. In general, barcodes are of sufficient length and comprise sequences that are sufficiently different to allow the identification of samples based on barcodes with which they are associated. In some aspects, a barcode, and the sample source with which it is associated can be identified accurately after the mutation, insertion, or deletion of one or more nucleotides in the barcode sequence. In some aspects, each barcode in a plurality of barcodes differs from every other barcode in the plurality at two or more nucleotide positions, such as at 2, 3, 4, 5, 6, 7, 8, 9, 10, or more positions. In some aspects, one or more adaptors comprise(s) at least one barcode sequence. For some disclosed aspects, the method comprises identifying the sample or source from which a target nucleic acid is derived based on a barcode sequence to which the target nucleic acid is joined, and / or identifying a target nucleic acid based on a barcode sequence to which the target nucleic acid is joined. Some aspects of the method further comprise molecular counting applications via digital barcode enumeration and / or binning to determine expression levels or copy number status of desired targets.

[0075] “Click chemistry” and “click reaction” are used interchangeably herein and are intended to be consistent with their use in the art. Generally, click chemistry reactions are fast, simple, easily purified, and regiospecific. Click chemistry includes reactions such as, but not limited to, copper catalyzed azide-alkyne cycloaddition (CuAAC); strain-promoted azide-alkyne cycloaddition (SPAAC), also known as copper-free click chemistry; strain-promoted alkyne-nitrone cycloaddition (SPANC); alkyne hydrothiolation; and alkene hydrothiolation. Click chemistry using copper as a catalyst often includes a Cu(I) stabilizing ligand that is labile. Without being bound by any particular theory, the ligand can stabilize or protect the Cu(I) ion from oxidizing from the reactive Cu(I) to Cu(II) and can also act as a proton acceptor reducing or eliminating requirement of a base in the reaction. Click chemistry between polynucleotides can, in some aspects, be assisted by using a moiety that brings the two reacting partners in close enough proximity to react.

[0076] “Complementary” refers to the broad concept of sequence complementarity between regions of two polynucleotide strands or between two nucleotides through base-pairing. For example, an adenine nucleotide is capable of forming specific hydrogen bonds (“base pairing”) with thymine or uracil. Similarly, a cytosine nucleotide is capable of base pairing with a guanine nucleotide.

[0077] The term “ddNTP” refers to 2’,3’-dideoxynucleotidetriphosphate, where the nucleotide comprises a native or non-native nucleobase. The term “dNTP” refers to 2’-deoxynucleotidetriphosphate, where the nucleotide comprises a native or non-native nucleobase.

[0078] “Double-stranded” when used in reference to a polynucleotide, means that some or all of the nucleotides between complementary strands of a polynucleotide are hydrogen bonded together, and may form a partial or complete double helix. A partially double stranded polynucleotide can have greater than 0% to at least 95% of its nucleotides are hydrogen bonded to complementary nucleotides, such as at least 10%, 25%, 50%, 60%, 70%, 80%, 90% or 95% of its nucleotides are hydrogen bonded to a complementary nucleotide.

[0079] “Handle” (interchangeably used with the terms “amplification handle” or “PCR handle”) refers to a functional component of an oligonucleotide sequence which itself is an oligonucleotide or polynucleotide sequence that provides an annealing site for amplification of the constructed oligonucleotide sequence. Handles used in the present aspects can be DNA, RNA, PNA, polymers comprising modified nucleotide bases modified bases or combinations of these bases, or polyamides, and the like.

[0080] “Library” when used in reference to nucleic acids, is intended to mean a collection of nucleic acids having different chemical compositions such as different sequence, different length, etc. The nucleic acids in a library likely will be different species and may have a common feature or characteristic of a genus or class, that otherwise differ in some way. For example, a library can include nucleic acid species that differ in nucleotide sequence, but that are similar with respect to having a sugarphosphate backbone. A library can be created using techniques known in the art. Nucleic acids exemplified herein can include nucleic acids obtained from any source, including, for example, digestion of a genome such as a human genome or a mixture of genomes. In another example, nucleic acids can be those obtained from metagenomic studies of a particular environment or ecosystem. The term also includes artificially created nucleic acid libraries such as DNA libraries.

[0081] “Labels” generally refers to chemical or biochemical moieties useful for labeling a nucleic acid. “Labels” include, for example, fluorescent agents, chemiluminescent agents, affinity agents, blocking groups, chromogenic agents, quenching agents, radionucleotides, enzymes, substrates, cofactors, inhibitors, nanoparticles, magnetic particles, and other moieties known in the art. Labels are capable of generating a measurable signal and may be covalently or non-covalently joined to an oligonucleotide or nucleotide. In some examples, an oligonucleotide or a portion of an oligonucleotide of oligonucleotide-tethered nucleotide as disclosed herein may serve as a label.

[0082] “Lectin” generally refers to a type of protein found in both plants and animals and are carbohydrate-binding proteins that are highly specific for their carbohydrate moieties. They may bind to a soluble carbohydrate or to a carbohydrate moiety which is a part of a glycoprotein or glycolipid. They typically agglutinate certain animal cells and / or precipitate glycoconjugates. Lectins are involved in biological recognition phenomena, such as cell-cell and cell-matrix interactions, cell growth and cell death, routing and migration, storage, signaling, environmental adaptation, defense mechanisms, etc.

[0083] “Linker” generally refers to a single covalent bond or a series of stable covalent bonds incorporating nonhydrogen atoms chosen selected from C, N, O, S and P. Exemplary linking members include at least one moiety selected from — C(O)NH — , — C(O)C) — , — NH — , — S — , — O — , alkyl, alkenyl, and alkynyl chains. A linker may also comprise a combination of 2 or more linking members.

[0084] “Nucleic acid synthesis” refers to any in vitro method for making a new strand of polynucleotide or elongating an existing polynucleotide such as DNA or RNA. Synthesis, according to the disclosure, includes amplification, which increases the number of copies of a polynucleotide template sequence using a polymerase. Polynucleotide synthesis incorporates nucleotides into a polynucleotide, for example via a primer to form a new polynucleotide molecule complementary to the polynucleotide template. The formed polynucleotide molecule and its template can be used as templates to synthesize additional polynucleotide molecules. Nucleic acid synthesis may also refer to reverse transcription, for example, of an RNA (such as an mRNA) to DNA (such as a cDNA).

[0085] “Oligonucleotide-tethered nucleotide” refers to a molecule, comprising two or more deoxyribonucleotides and / or ribonucleotides, that is covalently attached to a nucleotide nucleobase. For example, two or more deoxyribonucleotides and / or ribonucleotides are covalently attached through a triazole ring to a nucleotide nucleobase. Such covalent attachment may be a result of a “click chemistry” process. In some aspects, oligonucleotide-tethered nucleotides can be referred to as OTDN (oligonucleotide-tethered deoxynucleotide) or OTDDN (oligonucleotide-tethered di deoxy nucl eoti de) .

[0086] “Polyalkylene glycol” refers to straight or branched polyalkylene glycol polymers, such as polyethylene glycol, polypropylene glycol, and polybutylene glycol. Exemplary polyalkylene glycols have from 2 to 10 alkylene glycol units. The term “alkylene glycol subunit” refers to a single alkylene glycol unit. For example, an ethylene glycol subunit would be — O — CH2 — CH2 — . Exemplary polyalkylene glycols include polyethylene glycol having 2 to 10 ethylene glycol units, and in particular aspects, having 2, 4, or 6 ethylene glycol units, which are also referred to as PEG2, PEG4 and PEG6 respectively.

[0087] “Polynucleotide” refers to a polymer molecule comprising nucleotide monomers covalently bonded in a chain. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are examples of polynucleotides. Polynucleotides may be double stranded or single stranded, such as DNA, RNA, linear or circular dsDNA / ssDNA, fragmented dsDNA / RNA, linear or circular RNA, and other known polynucleotide forms.

[0088] “Polymerase” refers generally to an enzyme that catalyzes the reaction between 3 ’-OH and 5 ’-triphosphate in nucleotides, oligomers, and their analogs to form nucleic acid polymers. Polymerases include, but are not limited to, DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, RNA-dependent DNA polymerases, RNA-dependent RNA polymerases, template-independent DNA polymerase, templateindependent RNA polymerases, T7 DNA polymerase, T3 DNA polymerase, T4 DNA polymerase, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, DNA polymerase 1, KI enow fragment, Therm ophilus aquaticus DNA polymerase, Tth DNA polymerase, Phusion DNA Polymerase, SuperFi DNA Polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, Bst DNA Polymerase Large Fragment, Stoeffel Fragment, 9° N DNA Polymerase, Pfu DNA Polymerase, Tfl DNA Polymerase, Phi29 Polymerase, Tli DNA polymerase, eukaryotic DNA polymerase beta, telomerase, KOD HiFi, K0D1 DNA polymerase, Q-beta replicase, terminal transferase (TdT), AMV reverse transcriptase, M-MLV reverse transcriptase, Phi6 reverse transcriptase, HIV-1 reverse transcriptase, Thermo Sequenase (Thermo Fisher Scientific). These polymerases include wild-type, mutant isoforms, chimeric forms, and genetically engineered variants, such as exo-polymerases and other mutants, and incorporate them into a strand of nucleic acid.

[0089] “Primer” or “extension primer” refers to an oligonucleotide, whether occurring naturally or produced synthetically, that is capable of acting as a point of initiation of nucleic acid synthesis under appropriate conditions, for example, when in the presence of nucleotide triphosphates and a polymerase enzyme such as a thermostable polymerase enzyme, in an appropriate buffer and at a suitable temperature. The primer may be, in some aspects, single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. If double-stranded, the primer may be first treated to separate its strands before being used to prepare extension products. In some aspects, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products. The exact lengths of suitable primers will depend on many factors, including temperature, source of primer, the polymerase enzyme (for example, whether it is thermostable), and the method used. As used herein, the term “adapter” refers generally to any linear oligonucleotide that can be added, for example, ligated, to a nucleic acid molecule, thereby generating nucleic acid products that can be sequenced on a sequencing platform. In some aspects, adapters include two reverse complementary oligonucleotides that form a double-stranded structure.

[0090] “Single-stranded polynucleotide” refers to a polynucleotide or a portion thereof that has few to no hydrogen bonds with another polynucleotide, such that a double helix is not formed or is unstable under a given set of hybridization conditions.

[0091] “Splint oligonucleotide” refers to an oligonucleotide that is used as a template to facilitate the extension or ligation of nucleic acid sequences to an existing nucleic acid product in a template dependent manner to form an extended nucleic acid product, but that is not extended or incorporated into a nucleic acid product. By way of example only, in some aspects described herein, a splint oligonucleotide can comprise the following components: a sequence that enables hybridization to a handle sequence present in a nucleic acid product to be extended; a barcode sequence; and a sequence that is a template for the incorporation of a handle sequence different from the handle in into an extended nucleic acid product. In some aspects, splint oligonucleotides may also include a modification that prevents extension of the splint oligonucleotide from the 3’ end, which can be a 3’ amino, a 3’ phosphate, a 3’ dideoxy, or the like.

[0092] “Split-pool” generally refers to combinatorial barcoding to profile single cell transcriptomes by labeling the cellular origin of RNA compatible with fixed cells or nuclei from a sample and allows efficient sample multiplexing. In each split-pool round, fixed cells and / or nuclei are split (distributed) into containers and are labeled with well-specific barcodes, then pooled (extracted and combined). The fixed cells and / or nuclei from a sample are split, barcoded, and pooled multiple times. Thus, samples from a given cell will have a unique set of barcodes and / or combination of barcodes compared to the other single cells from the original sample.

[0093] “Template DNA molecule” generally refers to a nucleic acid strand from which a complementary nucleic acid strand is synthesized by a DNA polymerase in, for example, a primer extension reaction.

[0094] “Template dependent manner” generally refers to a process that involves the template-dependent extension of a primer molecule such as via DNA synthesis by DNA polymerase, cDNA synthesis by reverse transcriptase, or the like. The term “template dependent manner” typically refers to polynucleotide synthesis of RNA or DNA wherein the sequence of the newly synthesized polynucleotide strand is dictated by the well-known rules of complementary base pairing.

[0095] “Tag” or “oligonucleotide tag” refer to any nucleotide sequence added to the nucleic acid with oligonucleotide-tethered nucleotide. Non-limiting examples of such information include the addition of a component such as a scaffold or a building block, as in a scaffold tag or a building block tag, respectively; the headpiece in the library; the identity of the library (identity tag); the use of the library (use tag), and / or the origin of a library member (an origin tag). In some aspects the components are added via a binding reaction.

[0096] “Unique molecular indices” (UMIs) refers to the sequences of nucleotides applied to or identified in DNA molecules that can be used to distinguish individual DNA molecules from one another. Since UMIs are used to identify DNA molecules, they are also referred to as unique molecular identifiers. UMIs may be sequenced along with the DNA molecules with which they are associated to determine whether the read sequences are those of one source DNA molecule or another.

[0097] “Universal handle” refers to generic sequences suitable as an annealing site for extension by a polymerase as described elsewhere herein, a reverse transcriptase, a DNA polymerase, or the like, or contain extension primer binding sites, sequencing primer binding sites, or the like. In some aspects, the universal handles used in the aspects disclosed herein are about 10 of such monomeric components. In other aspects, the universal handle is at least about 5 to 100 monomeric components, for example, nucleotides, in length. Thus in various aspects, the universal handles described herein are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,

[0098] 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,

[0099] 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95,

[0100] 96, 97, 98, 99 or up to 100 monomeric components.

[0101] “Universal” sequence generally refers to an oligonucleotide, such as a primer, adapter, etc., comprising a known sequence, which may be for use as a primer or probe binding site using a primer or probe of a known sequence, which may be complementary to the universal sequence. While a template-specific sequence of a primer, a barcode sequence of a primer, and / or a barcode sequence of an adaptor might differ in aspects of the technology, which may be from fragment to fragment, from sample to sample, from source to source, or from region of interest to region of interest, a universal sequence is the same from fragment to fragment, from sample to sample, from source to source, or from region of interest to region of interest so that all fragments comprising the universal sequence can be handled and / or treated in a same or similar manner, for example, amplified, identified, sequenced, isolated, etc., using similar methods or techniques which may use the same primer or probe.

[0102] II. Functionalized Magnetic Particle Bound to a Permeabilized Cell

[0103] Certain aspects of the present disclosure concern a magnetic particle bound to a cell. The magnetic particle-cell construct can be used for a variety of purposes and is particularly useful for preparing nucleic acid libraries for Next Generation Sequencing. The outer surface of the magnetic particle has an affinity for a cell and can bind to a cell, including a permeabilized cell, allowing for oligonucleotides to label or tag a plurality of different types of nucleic acids within the cell, typically via multiple series of splitting and pooling. The multiple series of splitting and pooling allows for samples from a single cell to have a unique barcode or combination of barcodes compared to other single cells that were in the original sample. Additionally, multiple series of splitting and pooling allows for bulk processing of cells, is amenable to multiplexing, and to high-throughput processing.

[0104] In some aspects, provided are compositions including a functionalized magnetic particle comprising at least one ligand; at least one cell bound to the at least one ligand, wherein the cell comprises at least one cellular nucleic acid; and at least one nucleic acid complementary to the at least one cellular nucleic acid (such as a cDNA), wherein the complementary nucleic acid comprises a first barcode and an oligonucleotide tethered nucleotide.

[0105] In some aspects, the cell is fixed, such that nucleic acids and proteins within the cell remain intact and fixed within their cell origin. In additional aspects, the cell is permeabilized, such that reagents such as enzymes (e.g., polymerases), nucleotides, primers, and the like are able to enter the cells wherein reverse transcription reactions, primer extension reactions, ligation reactions, amplification reactions, and the like can occur.

[0106] In some aspects, the cell is a eukaryotic cell, such as a mammalian cell. For example, a cell may be derived from a human, monkey, mouse, cow, swine, goat, hamster, rat, cat, or dog, and the like. In some aspects, the cell is hematopoietic, such as an immune cell such as a cell of the innate or adaptive immune system including but not limited to a B cell, a T cell, a natural killer (NK) cell, a pluripotent stem cell, an induced pluripotent stem cell, a chimeric antigen receptor T (CAR-T) cell, a monocyte, a macrophage, and a dendritic cell.

[0107] In some aspects, the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mTMCD-3, NHDF, HeLaS3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL-2, CIR, Rat6, CVI, RPTE, A1O, T24, 182, A375, ARH-77, Calul, SW480, SW620, SK0V3, SK-UT, CaCo2, P388DI, SEM-K2, WEHI-231, HB56, TIB55, lurkat, 145.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4. COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB / 3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP- I cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1 / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-KI, CHO-K2, CHO-T, CHO Dhfr- / -, COR-L23, COR- L23 / CPR, COR-L235010, CORL23 / R23, COS-7, COV-434, CML TI, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT- 29, lurkat, 1Y cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCKII, MDCKI1, MOR / 0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCLH69 / CPR, NCI- H69 / LX10, NCI-H69 / LX20, NCLH69 / LX4, NIH-3T3, NALM-1, NW-145, OPCN / OPCT cell lines, Peer, PNT-1A / PNT 2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. In an exemplary aspect, the cell is a U937 cell (cell culture suspension cell, human monocyte). In another exemplary aspect, the cell is a PBMC cell (human peripheral blood mononuclear cell). In yet another exemplary aspect, the cell is a HEK 293 cell (cell culture adherent cell, immortalized human embryonic kidney cell). In another exemplary aspect, the cell is a 3T3 cell (cell culture adherent cell, mouse fibroblast).

[0108] Cells can be prepared as desired for a particular application, such as by using fixation agents and / or permeabilizing agents. In some aspects, the fixation agent can be, but is not limited to, formaldehyde, formalin, methanol, paraformaldehyde, methanol, acetic acid, SUPERase-In, PBS and the like. Agents for permeabilizing cells include, but are not limited to, triton 100, saponin, Tween 20, PBS, BSA, SUPERase-In, and organic solvents such as methanol, acetone and the like.

[0109] The magnetic particles disclosed herein can be ferromagnetic particles, paramagnetic particles, superparamagnetic particles, or mixtures of various classes of magnetic particles. Paramagnetism occurs in the presence of unpaired electrons in a material. Paramagnetic materials exhibit magnetism in the presence of an external magnetic field. In some aspects, the paramagnetic materials can include aluminum, oxygen, titanium, and iron oxide (FeO). Materials that are ferromagnetic can be magnetized by an external magnetic field and can remain magnetized after the external magnetic field is removed. Ferromagnetism is also of the material's crystalline structure and microstructure. For example, there are ferromagnetic metal alloys that are comprised of elements that are not ferromagnetic. Examples of ferromagnetic materials include iron, nickel, and cobalt. A ferrimagnetic material can have multiple populations of atoms with opposing magnetic moments. The crystal structure of a ferrimagnetic material comprises magnetic sublattices of magnetic moments, wherein the magnetic moments of the two sublattices are anti-aligned and not equal. When the external magnetic field is removed from a ferrimagnetic material, the ferrimagnetic material can become unmagnetized. An example of a ferrimagnetic material is a ferrite. Superparamagnetism is when nanoparticles, for example smaller than 50 nm in size, are made of a ferromagnetic or ferrimagnetic material and are small enough to contain a single magnetic domain. Superparamagnetic materials can exhibit paramagnetic-like behavior outside of a magnetic field and can be more magnetically responsive than paramagnetic materials in the presence of an external magnetic field. Furthermore, the magnetic particles are not attracted to one another when an external magnetic field is removed. As a result, the magnetic particles can remain suspended without magnetically induced aggregation occurring after mixing and thus do not inhibit binding or elution. Magnetic particles can be magnetically separated from a container using an extraction module, such as a magnet or an electromagnet, as described herein.

[0110] The magnetic particles can be any of a variety of shapes, and the shape can be selected to maximize the surface areas of the particles. In some aspects, the magnetic particles can be spherical, bar shaped, elliptical, or any other suitable shape. The magnetic particles can be a variety of densities, which can be determined by the composition of the core. In some examples, the density of the magnetic particles can be adjusted with a coating, as described herein. In some aspects, the magnetic particles have sufficient surface area to permit efficient binding of a target analyte and are further characterized by having surfaces which are capable of reversibly or irreversibly binding the target analyte.

[0111] Magnetic particles can be sized to facilitate separation from solution by applying a magnetic field by filtration. In addition, magnetic particles should not be so large that their surface area is minimized or that they are not suitable for nanoscale-to-microscale manipulation. Sizes can range from greater than 0 nm mean diameter to 1 mm mean diameter, from 100 nm mean diameter to about 100 pm mean diameter, from 5 nm mean diameter to 50 pm mean diameter, from 1 nm mean diameter to 1 pm mean diameter. In other aspects, the magnetic particles can be microparticles having a mean diameter greater than 1 pm mean diameter, but less than 100 pm mean diameter. In some aspects, the magnetic components of the particles can be magnetic nanoparticles, magnetic sub-micrometer particles, or magnetic micrometer particles.

[0112] The magnetic particles described herein can have many different structures and / or compositions. In some aspects, the magnetic particles may further comprise an outer surface comprising a polymer matrix, agarose, or silica matrix. In some aspects, the magnetic nanoparticles or magnetic particles are functionalized with ligands that are incorporated into the outer surface to increase the affinity towards cells. The outer surface of the magnetic particle may be functionalized with a ligand to interact with cells in a sample such that the interaction between the ligand and cell is maintained when a magnetic field is applied to the magnetic particle. Attaching ligands that target the cell surface allows for the cells to be used in downstream assays while attached to the surface of the magnetic particle.

[0113] In some aspects, the ligand is a lectin, which interacts with cells via interactions on the cell surface. Lectins can bind to sugars, glycoproteins, and glycolipids with high affinity. In some aspects, the lectins may comprise abrin, aggrecan, asialoglycoprotein receptor, calnexin, calrecticulin, CD22, CD33, CD94, collectin (mannan -binding lectin), Concanavalin A, galectin, Griffonia simplicifolia II agglutinin, legume lectin, mannose receptor, myelin-associated glycoprotein, N-acetylglucosamine receptor, phytohaemagglutinin, Pisum sativum agglutinin, pokeweed mitogen, ricin, selectin, sialoadhesin, soybean agglutinin, Ulex europaeus agglutinin-I, versican, and Viscum album agglutinin, among others. In some aspects, the ligand is a carbohydrate. The carbohydrate may comprise dextran, dextran-hydrogel, other dextran derivatives, chitin, chitosan, fochrysolaminarin, xylan, arabinoxylan, mannan, fucoidan, galactomannan, or derivatives thereof. In an exemplary aspect, the ligand on the outer surface of a magnetic particle is Concanavalin A. Exemplary, commercially available and suitable products include Con A-Coated Microparticles (BioMag™), Magnetic Concanavalin A Beads (Agarose) (antibodies-online.com), Concanavalin A beads (BangsLabs), and / or the like.

[0114] The oligonucleotides described herein can comprise a nucleotide linked to an oligonucleotide of from about 3 to about 100 nucleotides in length (e.g., an oligonucleotide tethered nucleotide). The oligonucleotides can be used to label or tag a plurality of different types of nucleic acids in a plurality of different applications with a known oligonucleotide, which can carry unique sequences and serve as a barcode for further use in downstream applications. As illustrated by FIG. 1, an oligonucleotide 110 and an enzyme 120 may be used as reaction components in a nucleic acid synthesis reaction such that the oligonucleotide 110 may be incorporated into a nucleic acid 130 within a cell 140. Cell 140 is bound to a magnetic particle 150 via a ligand 160. In some aspects, the cell 140 can be subsequently permeabilized to allow reaction components to cross the cell membrane 170 to incorporate an oligonucleotide 110 into the nucleic acid 130.

[0115] Oligonucleotide 110 can be incorporated into nucleic acid 130 via a synthesis reaction, for example, an extension reaction, an amplification reaction, a TdT reaction, or the like. For the synthesis reaction to occur, as depicted by FIG. 2, a ligand 210 attached to the surface of a magnetic particle 220 is bound to a permeabilized cell 250 such that the oligonucleotide 230 and enzyme 240 can cross the membrane of the permeabilized cell 250. This allows for the incorporation of various types of functional sequences 260 into the nucleic acid 270, such as sequencing adapters, promoter sequences, barcodes, unique molecular identifiers, handle sequences, and the like. The incorporation can be direct by providing the functional sequence. The incorporation can also be indirect, by providing sequences that enable the addition of various functional sequences, as described further herein. Additionally, the permeabilized cells bound to the magnetic particle allow for permeabilized cells to be split, barcoded, pooled, and washed multiple times, resulting in samples from a single cell having a unique barcode, set of barcodes, or combinations of barcodes. Moreover, the barcoding scales directly with the number of available barcodes, allows for bulk cell processing, and is amenable to multiplexing, and high throughput automation.

[0116] Oligonucleotides may include a 5' end and a 3' end. The 5' end of an oligonucleotide may be a hydroxyl, a hydrophobic moiety, a 5' cap, a phosphate, a diphosphate, a triphosphate, a phosphorothioate, a diphosphorothioate, a triphosphorothioate, a phosphorodithioate, a diphosphrodithioate, a triphosphorodithioate, a phosphonate, a phosphoramidate, or a neutral organic polymer. The 3' end of an oligonucleotide can be a hydroxyl, a hydrophobic moiety, a phosphate, a diphosphate, a triphosphate, a phosphorothioate, a diphosphorothioate, ta riphosphorothioate, a phosphorodithioate, a di sphorodi thioate, a triphosphorodithioate, a phosphonate, a phosphoramidate, or a neutral organic polymer. An oligonucleotide having a 5 '-hydroxyl or 5 '-phosphate has an unmodified 5' terminus. An oligonucleotide having a 5' terminus other than 5 '-hydroxyl or 5 '-phosphate has a modified 5' terminus. An oligonucleotide having a 3 '-hydroxyl or 3 '-phosphate has an unmodified 3' terminus. An oligonucleotide having a 3' terminus other than 3 '-hydroxyl or 3 '-phosphate has a modified 3' terminus. Oligonucleotides can be in double- or single- stranded form. Double-stranded oligonucleotide molecules may include one or more single-stranded segments such as overhangs. The covalent linkage between adjacent nucleosides in an oligonucleotide can form an intemucleoside linkage.

[0117] An internucleoside linkage is an unmodified internucleoside linkage or a modified intemucleoside linkage. An unmodified internucleoside linkage is a phosphate ( — O — P(O)(OH) — O — ) intemucleoside linkage (phosphate phosphodiester). A modified intemucleoside linkage is an intemucleoside linkage other than a phosphate phosphodiester. The two main classes of modified intemucleoside linkages are defined by the presence or absence of a phosphorus atom. Non-limiting examples of phosphorus-containing intemucleoside linkages include phosphodiester linkages, phosphotriester linkages, phosphorothioate diester linkages, phosphorothioate triester linkages, morpholino intemucleoside linkages, methylphosphonates, and phosphoramidate. Non-limiting examples of non-phosphorus intemucleoside linkages include methylenemethylimino, thiodiester, thionocarbamate, siloxane, and N,N'- dimethylhydrazine. Phosphorothioate linkages are phosphodiester linkages and phosphotriester linkages in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. In some aspects, an intemucleoside linkage is a group represented by Formula I z

[0118] _6_x— y — x-&-

[0119] 4

[0120] Formula I where Z is O, S, or Se; Y is — X-L-R1; each X is independently — O — , — S — , — N(-L- R1) — , or L; each L is independently a covalent bond or a linker such as a linker consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 monomers independently selected from the group consisting of a substituted C1-12 alkylene, a substituted C2-12 heteroalkylene, a substituted Ce-io arylene, a substituted C3-8 cycloalkylene, a substituted C1-9 heteroarylene, a substituted C1-9 heterocyclylene, — O — , — S — S — , and — NRN— , where each RNis independently H or substituted C1.12 alkyl); each R1is independently hydrogen, — S — S — R2, — O — CO — R2, — S — CO — R2, substituted C1.9 heterocyclyl, or a hydrophobic moiety; and each R2is independently substituted Ci-io alkyl, substituted C 2-10 heteroalkyl, substituted Ce-io aryl, substituted Ce-io aryl C1-6 alkyl, substituted C1.9 heterocyclyl, or substituted C1.9 heterocyclyl C1-6 alkyl.

[0121] When L is a covalent bond, R1is hydrogen, Z is oxygen, and all X groups are — O — , the internucleoside group is known as a phosphate phosphodiester. When L is a covalent bond, R1is hydrogen, Z is sulfur, and all X groups are — O — , the internucleoside group is known as a phosphorothioate diester. When Z is oxygen, all X groups are — O — , and either L is a linker or R1is not a hydrogen, the internucleoside group is known as a phosphotriester. When Z is sulfur, all X groups are — O — , and either L is a linker or R1is not a hydrogen, the intemucleoside group is known as a phosphorothioate tri ester.

[0122] The nucleobase is a nitrogen-containing heterocyclic ring found at the 1' position of the ribofuranose / 2'-deoxyribofuranose of a nucleoside. Nucleobases are unmodified or modified. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine and guanine, and the pyrimidine bases thymine, cytosine, and uracil. Modified nucleobases include 5-substituted pyrimidines, 6- azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6 substituted purines, as well as synthetic and natural nucleobases, for example, 5 -methyl cytosine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-alkyl (for example 6-methyl) adenine and guanine, 2-alkyl (for example, 2-propyl) adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5- halouracil, 5-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 5 -tri fluoromethyl uracil, 5 -trifluoromethyl cytosine, 7-methyl guanine, 7-methyl adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3 -deazaguanine, 3 -deazaadenine. Certain nucleobases are particularly useful for increasing the binding affinity of nucleic acids, e.g., 5-substituted pyrimidines; 6-azapyrimi dines; N2-, N6-, and / or 06-substituted purines. Nucleic acid duplex stability can be enhanced using, e.g., 5-methylcytosine. Non-limiting examples of nucleobases include: 2-aminopropyladenine, 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5- propynyl ( — C=C — CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6- azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5- bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7- methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3- deazaguanine, 3 -deazaadenine, 6 -N-benzoyl adenine, 2-N-isobutyrylguanine, 4-N- benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N- benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as l,3-diazaphenoxazine-2-one, l,3-diazaphenothiazine-2-one and 9- (2-aminoethoxy)-l,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deazaadenine, 7-deazaguanine, 2-aminopyridine, or 2- pyridone, and the like.

[0123] The nucleoside may comprise a sugar-nucleobase compound or modified or unmodified 2'-deoxyribofuranose-nucleobase compounds. The sugar may be ribofuranose. The sugar may be modified or unmodified. An unmodified ribofuranose- nucleobase is ribofuranose having an anomeric carbon bond to an unmodified nucleobase. Unmodified ribofuranose-nucleobases are adenosine, cytidine, guanosine, and uridine. Unmodified 2'-deoxyribofuranose-nucleobase compounds are 2'- deoxyadenosine, 2'-deoxycytidine, 2'-deoxyguanosine, and thymidine. The modified compounds and groups include one or more modifications selected from the group consisting of nucleobase modifications and sugar modifications described herein. A nucleobase modification is a replacement of an unmodified nucleobase with a modified nucleobase. A sugar modification may be, e.g., a 2'-substitution, locking, carbocyclization, or unlocking. A 2'-substitution is a replacement of 2'-hydroxyl in ribofuranose with 2'-fluoro, 2’-methoxy, or 2'-(2-methoxy)ethoxy. Alternatively, a 2'- substitution may be a 2'-(ara) substitution, which corresponds to Formula II

[0124] Formula II, where NB is a nucleobase, and R is a 2'-(ara) substituent (e.g., fluoro). 2'-(ara) substituents and can be same as other 2'-substituents described herein. In some aspects, 2'-(ara) substituent is a 2'-(ara)-F substituent (R is fluoro). A locking modification is an incorporation of a bridge between 4'-carbon atom and 2'-carbon atom of ribofuranose. Nucleosides having a locking modification such as bridged nucleic acids, e.g., locked nucleic acids (LNA), ethylene-bridged nucleic acids (ENA), and cEt nucleic acids. The bridged nucleic acids are typically used as affinity enhancing nucleosides.

[0125] Nucleotides comprise a nucleoside bonded to an intemucleoside linkage or a monovalent group of the following structure — X1— P(X2)(R1)2, where X1is O, S, or NH, and X2is absent, =0, or =S, and each R1is independently — OH, — N(R2)2, or — O — CH2CH2CN, where each R2is independently alkyl, or both R2groups, together with the nitrogen atom to which they are attached, combine to form a substituted heterocyclyl.

[0126] In some aspects, a nucleotide is linked to an oligonucleotide such that a polymerase incorporates an oligonucleotide-tethered nucleotide into a nucleic acid strand to provide a new priming site for nucleic acid synthesis initiation as described in U.S. Patent Application No. 16 / 908,567 filed on June 22, 2020, which is hereby incorporated by reference in its entirety. For example, two or more deoxyribonucleotides and / or ribonucleotides are covalently attached through a triazole ring to a nucleotide nucleobase. Such covalent attachment may be a result of a click chemistry process. The oligonucleotide-tethered nucleotides can be referred to as OTDN (oligonucleotide-tethered deoxynucleotide) or OTDDN (oligonucleotide- tethered dideoxynucleotide). A primer at least partially complementary to the tethered oligonucleotide is provided and allowed to anneal to the tethered oligonucleotide. The polymerase can extend the annealed primer through an unnatural linker on the oligonucleotide-tethered nucleotide, thereby generating a new nucleic acid strand.

[0127] In other aspects, a polymerase incorporates the oligonucleotide-tethered nucleotide into a nucleic acid strand to provide a universal handle sequence for annealing a splint oligonucleotide. The splint oligonucleotide includes a sequence capable of annealing to the universal handle sequence of the tethered nucleotide and a template for a desired sequence. The polymerase can extend the 3’ OH of the tethered oligonucleotide across the annealed splint oligonucleotide via template-directed polymerization, thereby incorporating any desired sequence. For example, barcodes, unique molecular identifiers, universal sequences, random sequences, unique molecular identifiers, promoters, and the like can be incorporated, thereby generating a new nucleic acid strand. The resulting new nucleic acid strand may be further manipulated using subsequent amplification, extension, ligation, or other treatments. By way of example, the resulting new nucleic acid strand can be amplified and / or subjected to an adapter addition reaction to provide a next generation sequencing library. The sequencing library is useful in numerous sequencing methods, and across a variety of platforms.

[0128] In some aspects, the oligonucleotide may be tethered to dNTP via the dNTP nucleobase. When an oligonucleotide is tethered to a dNTP, multiple incorporations of the nucleotide into a nucleic acid are possible. Alternatively, the oligonucleotide can be tethered to ddNTP via the ddNTP nucleobase. In aspects wherein the oligonucleotide is tethered to a ddNTP, incorporation of the oligonucleotide-tethered nucleotide terminates the nucleic acid synthesis.

[0129] In some aspects, the oligonucleotide-tethered nucleotides can be prepared using “Click” chemistry. For example, the oligonucleotide may be tethered to the nucleobase of a dNTP or ddNTP as a result of a click reaction, such as a (3+2) cycloaddition reaction between azide and alkyne groups, which forms a 1,2, 3 -triazole ring that chemically joins the oligonucleotide and the dNTP or ddNTP.

[0130] In some aspects, the tethered oligonucleotide can be used as a priming site for nucleic acid synthesis by nucleic acid polymerases. A primer complementary to the tethered oligonucleotide may have tailed sequences, which are used for adding adapters and / or barcodes for sequencing such as P5 and P7 sequences used to hybridize to Illumina flow cell.

[0131] In the aspects described herein, the oligonucleotide-tethered nucleotide can be modified with an affinity tag to facilitate target product enrichment.

[0132] Various nucleic acid polymerases have the ability to incorporate modified nucleotides bearing bulky groups attached to their nucleobases and it was expected that such modified nucleotides might be incorporated into the growing nucleic acid strand by nucleic acid polymerases during the nucleic acid copying process, initiated for instance from randomized hexamers, or would be added to the very 3’ end of single- or double-stranded nucleic acid by template-independent polymerases, such as terminal transferases. When modified nucleotides have a 3 ’-hydroxyl group on their sugar moiety, at least one and / or multiple incorporations of the oligonucleotide-bearing nucleotide into the copied nucleic acid are expected. Having multiple priming sites on such newly synthesized nucleic acid would, for example, facilitate isothermal amplification using both random hexamers and oligonucleotides which are complementary to the tethered oligonucleotides attached to incorporated nucleotides as extension primers. Oligonucleotide-bearing nucleotides incorporated at 3’ termini into the structure of double- stranded DNA with terminal transferase (TdT) may be used to add the fully or partially complementary oligonucleotide to the opposite DNA strand (complementary and mismatched adapter regions). By using appropriately designed oligonucleotides it is possible to generate DNA ends that are compatible with sequencing on various platforms, including but not limited to the Illumina platform.

[0133] While not being bound by a theory of operation, it is believed that efficient incorporation of modified nucleotides during nucleic acid synthesis is highly dependent on the size of an attached label. For example, the length of the linking group (or groups) between a nucleotide heterocyclic base and label may have significant impact on incorporation. The linker should be long enough to reduce label steric hindrance and changes of nucleotide steric structure. At the same time, it should be short enough to avoid back-folding onto the nucleic acid strand. Moreover, the terminal functional groups of the linker must be tolerated by nucleic acid polymerase enzymes. A properly designed linker will allow incorporation of nucleotides bearing large labels.

[0134] When the oligonucleotide-tethered nucleotide has a 3’-H instead of a 3’- hydroxyl group (for example, a dideoxy-modified nucleotide), incorporation of such oligonucleotide-tethered nucleotide would terminate the nucleic acid synthesis. When an oligonucleotide-tethered dideoxynucleotide (OTDDN) is used in synthesis reactions, a set of randomly terminated fragments is generated. By adjusting the concentration of the OTDDN in the synthesis reaction, for example, relative to the corresponding native nucleotides, the synthesis (and length of the synthesized strand) can be manipulated. In some aspects, the synthesis reaction includes a single type of OTDDN. The OTDNN may be an OTddATP, OTddTTP, OTddCTP, OTddCTP, OTddUTP. In some aspects, the synthesis reaction may include a combination of two or more OTDDNs, such as OTddTTP and OTddCTP, or other combinations. In some aspects, when there is a single type of OTDDN present in the reaction and accompanied by other, native nucleotides, the reaction does not contain for example, the corresponding native nucleotide. In some aspects, when there is a single type of OTDDN present in the reaction, the reaction contains relatively more OTDDN compared to the corresponding native nucleotide, about equal amounts of the OTDDN and corresponding native nucleotide, or relatively less OTDDN present, compared to the corresponding native nucleotide.

[0135] The resulting nucleic acids from an extension reaction utilizing an OTDDN can be further manipulated. For example, such further manipulations can include from an extension primer hybridized to the tethered oligonucleotide, or from extension of the tethered oligonucleotide across a splint oligonucleotide. Additionally, the resulting nucleic acids from an amplification reaction utilizing an OTDDN can be further manipulated using, for example, subsequent amplification, extension, ligation, or other treatments. The extension products that incorporate an OTDDN can then be subjected to downstream manipulations for further extension reactions, amplification reactions, and the like. In some examples, the extension products with incorporated OTDDN can be used in downstream extension or amplification (PCR) reactions for platform- specific, full-length sequencing adaptor introduction. In some aspects, this method can also be used to overcome the need for nucleic acid fragmentation.

[0136] The oligonucleotide-tethered nucleotides described herein may comprise affinity labels (for example, biotin-modified) to facilitate enrichment. Alternatively, the oligonucleotide-tethered nucleotides may comprise other labels.

[0137] In some aspects, the oligonucleotide-tethered nucleotides used herein generally have a structure represented by Formula III

[0138] Formula III, or a salt thereof wherein NB is a nucleobase; “Oligo” is a oligonucleotide of 3 to 100 nucleotides; X and Q are independently selected from, H, OH, N3, halo, alkyl , alkoxy, alkyl, alkenyl, alkynyl, acyl, cyano, amino, ester, and amido; Z and Y are independently selected from a bond, amino, amido, alkylene, alkenylene, alkynylene, thioether, sulfonyl, sulfonamido, ether, ketone, carbonyl, anhydride, ester, imido, urea, urethane, and combinations thereof; and CXN is selected from alkylene, alkenylene, alkynylene, ketone, carbonate, ester, ether, anhydride, amido, amino, aminoalkyl, imino, imido, diazo, carbamate ester, phosphodiester, sulfide, disulfide, sulfonyl, sulfonamido, and a heterocyclic group containing from one to four N, O, S atom(s) or a combination thereof. In some aspects, the salt of the compound of Formula III is a quaternary ammonium salt. In some aspects, X is selected from H, OH, F, N3, and amino. In other aspects, X is selected from H, N3, and OH. In some aspects, X is OH. In other aspects, X is H. In some aspects, Q is H, OH, F, Cl, Br, I, -N3. In other aspects, Q is H. In some aspects, X and Q are H. The oligonucleotide (“Oligo”) may be tethered to the nucleotide either through a 5 ’-phosphate to the nucleobase of the dNTP or ddNTP having Formula IV, or through a nucleobase (NB2) having Formula V

[0139]

[0140] Formula IV and Formula IV provide a more detailed view of how an Oligo of Formula III is tethered to the dNTP or ddNTP, wherein Oligo' of Formula IV and V represents all but one unit of the original oligo sequence represented by Formula III. “CXN” is a group formed by a reaction between functional groups on intermediates that couples the intermediates to form the oligonucleotide-tethered nucleotides disclosed herein. In other aspects, the reaction can form a heterocyclic group containing from one to four N, O, S atom(s) or a combination thereof where heterocyclic group is substituted at the carbon, nitrogen, or sulfur atom(s). In some aspects the reaction can form a heterocyclic group selected from: 5- membered heterocycles having one hetero atom, for example, pyrroles, thiophenes, furans, pyrrolidine, thiolane, tetrahydrofuran; 5- membered heterocycles bearing two heteroatoms at 1,2 or 1,3 positions (for example, isoxazoles, oxazoles, pyrazoles, imidazoles, isothiazoles, thiazoles); 5-membered heterocycles bearing three heteroatoms (for example, triazoles, oxadiazoles, thiadiazoles); 6-membered heterocycles bearing one heteroatom (for example, pyrans, thiopyrans, pyridines, tetrahydropyrans, tetrahydrothiopyrans, piperidines); 6-membered heterocycles bearing two heteroatoms (for example, pyridazines, pyrimidines, pyrazines, hexahydropyridazines, hexahydropyrimidines, piperazines, dioxanes, morpholines, thiazines, oxazines, dithianes); and 6- membered heterocycles bearing three heteroatoms (for example, triazines, dithiazines, thiadiazines, triazinanes, oxathiazines).

[0141] In some aspects, CXN of Formula III is selected from 5-membered heterocycles and 6-membered heterocycles each having from 1 to 3 heteroatoms. In some aspects, CXN is selected from pyrrolo, thiophenyl, furanyl, pyrrolidinyl, thiolanyl, tetrahydrofuranyl, isoxazolyl, oxazolo, pyrazolo, imidazolyl, isothiazolo, thiazolyl, triazolo, oxadiazolo, thiadiazolo, pyranyl, thiopyranyl, pyridinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, pyridazinyl, pyrimidinyl, pyrazinyl, hexahydropyridazinyl, hexahydropyrimidinyl, piperazinyl, dioxanyl, morpholino, thiazinyl, oxazino, dithianyl, triazinyl, dithiazino, thiadiazino, triazinanyl, and oxathiazino.

[0142] In some aspects, CXN is represented by Formula VI or Formula VII

[0143] Formula VI

[0144] Formula VII.

[0145] In some aspects, Z and Y of Formula III are each linkers, or linking moi eties, which refers to certain functional groups that may be varied to provide the overall tether between the ddNTP or dNTP and the oligonucleotide with the desired properties. In some aspects each of Z and Y is independently selected from a bond, amino, amido, alkylene, alkenylene, alkynylene, thioether, sulfonyl, sulfonamido, ether, ketone, carbonyl, anhydride, ester, imido, urea, urethane, and combinations thereof. In some aspects, each Z and Y are independently chosen from amino, amido, alkylene, alkenylene, alkynylene, ether, ketone, carbonyl, anhydride, ester, imide, or any combination thereof. In other aspects, Y is alkylene or alkynylene. In other aspects, Z is a combination of one or more of alkynylene, alkylene, ether and amido. In a particular aspect, Z is

[0146] -(CH2-CH2)C(O)(CH2CH2)NHC(O)(CH2)5- or -HN-.

[0147] In still other aspects, the combination of -NB-Z- is represented by Formula VIII, ,

[0148] NB-HN-Li-, or -NB-(CH-CH)C(O)(CH2CH2)NHC(O)-LI, where Li is selected from alkylene, alkenylene, alkynylene, and polyalkylene glycol.

[0149] In some aspects, compounds satisfying Formula III may also satisfy one or more of Formula X - Formula XIII:

[0150] Formula X; Formula XIII; and salts thereof, wherein Oligo is the remaining 2 to 99 nucleotides from the Oligo group and NB2 is a nucleobase; Li is selected from alkylene, alkenylene, alkynylene, and polyalkylene glycol; L2 is alkylene or alkynylene. In some aspects CXN includes one or more of the groups recited herein that may be made using click chemistry. In some aspects the linking group Z of Formula III comprises the subgroup Li, which is selected from alkylene, alkenylene, alkynylene, and polyalkylene glycol. In other aspects, Li is C2-C 12 alkylene, C2-C12 alkenylene, C2-C12 alkynylene, and polyalkylene glycol having from 2 to 8 glycol units. In other aspects, Li is selected from polyethylene glycol with 2 glycol units (PEG2), polyethylene glycol with 4 glycol units (PEG4), or polyethylene glycol with 6 glycol units (PEG6), methylene, ethylene, n-propylene, isopropylene, 1-butylene, cis-2-butylene, trans-2 -butylene, isobutylene, 1- pentylene, cis-2-pentylene, trans-2 -pentylene, isopentylene, and hexylene. In yet other aspects, Li is selected from -CH2-, -(CH2)3-, -(012)5-, PEG2, and PEG4.

[0151] In some aspects the linking group Y comprises L2 subgroup, which is selected from alkylene or alkynylene. In other aspects L2 is C2-C12 alkylene or C2-C12 alkynylene. In other aspects the combination of L2-Oligo is selected from -(012)4- Oligo or - (CH2)4C=C-Oligo. The Oligo may be tethered to the nucleotide either through a 5 ‘-phosphate to the nucleobase of the dNTP or ddNTP, or through a nucleobase (NB2).

[0152] NB and NB2 are independently a nucleobase. In some aspects the nucleobase is selected from adenine, 7-deazaadenine, cytosine, guanine, 7-deazaguanine, thymine, uracil and inosine. NB2 is a single nucleotide of the Oligo group so that the combination of NB-Oligo is an oligo. In some aspects, NB is a pyrimidine, and the pyrimidine is tethered to the oligonucleotide at the 5 position of the nucleobase. In other aspects, NB is a purine tethered to the oligonucleotide at the 7 position of the nucleobase.

[0153] The oligonucleotide-tethered nucleotides used herein generally have a structure according to Formula XIV

[0154] Formula XIV, or a salt thereof, wherein X is H, OH or N3, NB represents a nucleobase, Z and Y are linkers, Oligo represents an oligonucleotide of 3 to 100 nucleotides in length and Click represents the product of a Click reaction, which covalently binds the Z and Y linkers. In some aspects, the nucleobase is selected from adenine, 7-deazaadenine, cytosine, guanine, 7-deazaguanine, thymine, uracil, and inosine. In some aspects, Z and Y each independently comprise at least one linking moiety selected from a bond, amino, amido, alkyl, alkenyl, alkynyl, thioether, sulfonyl, sulfonamido, ether, ketone, carbonyl, anhydride, ester, imide, urea, urethane, or any combination thereof, or any combination thereof.

[0155] Alternatively, the oligonucleotide tethered nucleotide can be acyclic as represented by Formula XV

[0156] Formula XV, where the structural variables are as stated for Formula XIV.

[0157] The Click reaction product includes the products of reactions such as, but not limited to, copper catalyzed azide-alkyne cycloaddition (CuAAC); strain-promoted azide-alkyne cycloaddition (SPAAC), also known as copper-free click chemistry; strain-promoted, alkyne-nitrone cycloaddition (SPANC); alkyne hydrothiolation; and alkene hydrothiolation.

[0158] In some aspects, the Click reaction is a (3+2) cycloaddition reaction of an azide and an alkyne, forming a 1,2,3-triazole, and providing an oligonucleotide-tethered nucleotide according to Formula XVI,

[0159] Formula XVI, or a salt thereof, wherein, X, NB, Z, Y and Oligo are as defined above. In Formula XVI, one of Z and Y is covalently bound to the 1 position of the triazole, while the other of Z and Y is covalently bound to the 4 or 5 position of the triazole. In one aspect, X is OH; in another aspect X is H; and in yet another aspect X is N3. In some aspects, the linkers Z and / or Y include a carbon-based chain, for example an alkyl chain having 1 to 12 carbon atoms that may be linear or branched. In some aspects, the alkylene is a straight or branched Ci-Ce alkylene. Linkers Z and / or Y may also include a straight or branch alkenylene having 2 to 12 carbon atoms. Alternatively, the alkenylene is a straight or branched Ci-C alkenylene. In some aspects, linkers Z and Y include a straight or branched alkynylene chain of 2 to 12 carbons. In some aspects, the alkynylene is a straight or branched C2 to Ce alkynylene.

[0160] In some aspects, Z and / or Y includes a polyalkylene glycol having from 2 to 20 alkylene glycol units, while in other aspects, the polyalkylene glycol has 2 to 8 alkylene glycol units. In some aspects, the polyalkylene glycol has 2, 4, or 6 to 8 glycol units. Suitable alkylene glycol units include ethylene glycol, 1,2-propane-diol, 1,2-butylene glycol, and the like.

[0161] The oligonucleotide-tethered nucleotide may more particularly have a structure represented by Formula XVII

[0162] Formula XVII, or a salt thereof. Li and L2 are each linkers independently comprising an alkylene, an alkynylene, a polyalkylene glycol, or any combination thereof.

[0163] In some aspects, the oligonucleotide-tethered nucleotide may have the structure of Formula XVII, or a salt thereof, wherein Li is a linker comprising an alkylene, a polyalkylene glycol, or a combination thereof, and L2 is a linker comprising an alkynylene having from 2 to 12 carbons. More particularly, L2 is hexynyl. The polyalkylene glycol may be a polyethylene glycol having from 2 to 6 ethylene glycol units. In another aspect, Li comprises an alkylene having 1 to 12 carbon atoms.

[0164] More particularly, the alkylene is methylene, ethylene, n-propylene, isopropylene, 1- butylene, cis-2-butylene, trans-2-butylene, isobutylene, 1-pentylene, cis-2 -pentylene, trans-2-pentylene, isopentylene, or hexylene.

[0165] Alternatively, when strain-promoted, azide-alkyne cycloaddition (SPAAC) is used to generate oligonucleotide- tethered nucleotides, the resulting oligonucleoti detethered nucleotides described herein generally have a structure according to Formula

[0166] XVIII

[0167] Formula XVIII, or a salt thereof, wherein X is H or OH or N3, NB represents a nucleobase, Z and Y are linkers, and Oligo represents an oligonucleotide of 3 to 100 nucleotides in length. In some aspects, the nucleobase is selected from adenine, 7-deazaadenine, cytosine, guanine, 7-deazaguanine, thymine, uracil, and inosine. In some aspects, Z and Y each independently comprises at least one linking moiety selected from amino, amido, alkyl, alkenyl, alkynyl, thioether, sulfonyl, sulfonamido, ether, ketone, carbonyl, anhydride, ester, imide, urea, urethane, or any combination thereof.

[0168] In certain alternative aspects, azide modification can be introduced at the 3’ position of the nucleotide and therefore the oligonucleotide can be covalently tethered to the 3’ position of the nucleotide, resulting in Formula XIX and Formula XX O O O HO- -O-P-O- -O

[0169] 6H AH 611

[0170] Click Oligo Formula XIX

[0171] Formula XX, wherein NB represents a nucleobase, Y is a linker, Oligo represents an oligonucleotide of 3 to 100 nucleotides in length. In some aspects, the nucleobase is selected from adenine, 7-deazaadenine, cytosine, guanine, 7-deazaguanine, thymine, uracil and inosine. In some aspects, Z and Y each independently comprises at least one linking moiety selected from — C(0)NH — , — C(O)C) — , — NH — , — S — , — O — , alkyl, alkenyl, and alkynyl, or any combination thereof.

[0172] The oligonucleotide-tethered nucleotide of the present disclosure comprises, in some aspects, a pyrimidine nucleobase. In these aspects, the pyrimidine nucleobase is bound to the oligonucleotide at the 5 position of the pyrimidine, as represented by

[0173] Formula XXI.

[0174] Alternatively, when the oligonucleotide-tethered nucleotide comprises a purine base, the purine nucleobase is bound to the oligonucleotide at the 7 position of the nucleobase. In other aspects, an oligonucleotide-tethered nucleotide is obtained using oligonucleotide with C6(hexynyl)-amido moiety on a phosphate group at the 5’ end according to Formula XXII

[0175] Formula XXII.

[0176] Salts of the oligonucleotide-tethered nucleotides of the present disclosure include quaternary ammonium salts, sodium salts, potassium salts and the like.

[0177] In some aspects, the oligonucleotide of the oligonucleotide tethered nucleotide, is tethered to the nucleotide at its 5’ end. In some aspects, alkyne modification is added to the oligonucleotide nucleobase via a spacer of 8 carbon atoms or alternatively the alkyne group is attached to the phosphate of the 5’ terminus of the oligonucleotide via hexynyl linker. In some examples, the oligonucleotide has a modification, for example, at its 3’ end. In some examples, the modification is biotin, phosphate, amine or phosphorothioate modifications.

[0178] In some aspects, the oligonucleotide of an oligonucleotide-tethered nucleotide comprises deoxyribonucleotides. In some aspects, the oligonucleotide of an oligonucleotide-tethered nucleotide comprises ribonucleotides. In some aspects, the oligonucleotide of an oligonucleotide-tethered nucleotide comprises deoxyribonucleotides and ribonucleotides.

[0179] The length of a tethered oligonucleotide varies, and may depend on how the oligonucleotide-tethered nucleotide is used, as will be understood by a person of ordinary skill in the art. In some examples, the oligonucleotide is from 3 to 100 nucleotides, from 10 to 100 nucleotides, from 10 to 50 nucleotides, or from 20 to 40 nucleotides. The oligonucleotides of the oligonucleotide-tethered nucleotides described herein is not limited to any specific sequence. Rather, the oligonucleotide of the oligonucleotide-tethered nucleotides described herein may comprise a barcode sequence, an adapter sequence, a unique molecular identifier, an index sequence, an annealing site for polymerases, a handle sequence, a universal handle, a universal sequence, or the like, a random sequence, a target-specific sequence, or any combination thereof.

[0180] In some examples, oligonucleotide-tethered nucleotides are selected from compounds of Formula X and Formula XII, where X is OH, Q is H, Li is Ci alkylene, C3 alkylene, C5 alkylene, polyethylene glycol with 2 glycol units (PEG2), or polyethylene glycol with 4 glycol units (PEG4), and L2 is C1-C12 alkylene or C1-C12 alkynylene. In some examples, the combination of L2-Oligo is (CH2)4C=C-Oligo.

[0181] In some examples, oligonucleotide-tethered nucleotides are selected from compounds of Formula XI and Formula XIII, where X is OH, Q is H, Li is Ci alkylene, C3 alkylene, C> alkylene, polyethylene glycol with 2 glycol units (PEG2), or polyethylene glycol with 4 glycol units (PEG4), and L2 is C1-C12 alkylene or C1-C12 alkynylene. In some examples, the combination of L2-Oligo is -(CH2)4-Oligo.

[0182] In some examples, oligonucleotide-tethered nucleotides are selected from compounds of Formula X and Formula XII, where X is H, Q is H, Li is Ci alkylene, C3 alkylene, C5 alkylene, polyethylene glycol with 2 glycol units (PEG2), or polyethylene glycol with 4 glycol units (PEG4), and L2 is C1-C12 alkylene or C1-C12 alkynylene. In some examples, the combination of L2-Oligo is (CFFEOC-Oligo.

[0183] In some examples, oligonucleotide-tethered nucleotides are selected from compounds of Formula XI and Formula XIII, where X is H, Q is H, Li is Ci alkylene, C3 alkylene, C5 alkylene, polyethylene glycol with 2 glycol units (PEG2), or polyethylene glycol with 4 glycol units (PEG4), and L2 is C1-C12 alkylene or C1-C12 alkynylene. In some examples, the combination of L2-Oligo is -(CH2)4-Oligo.

[0184] Further exemplary oligonucleotide-tethered nucleotides are selected from compounds of Formula X and Formula XII, where X is H, Q is H, Li is Ci alkylene, the combination of L2-Oligo is (CFF^OC-Oligo. and NB and NB2 are independently selected from thymine, adenine, guanine, cytosine, or uracil. Further exemplary oligonucleotide-tethered nucleotides are selected from compounds of Formula XI and Formula XIII, where X is H, Q is H, Li is Ci alkylene, the combination of L2-Oligo is - (CH2)4-Oligo, NB and NB2 are independently selected from thymine, adenine, guanine, cytosine, or uracil.

[0185] The following list provides additional representative oligonucleotide-tethered nucleotides.

[0186] Formula XXIV

[0187]

[0188] Formula XXVI

[0189]

[0190] Formula XXIX Formula XXXII Formula XXXV

[0191] The oligonucleotide-tethered nucleotides used to practice disclosed aspects of the present disclosure may be used at suitable concentration such as a concentration of from 1 fmol to at least 10 pmol. In certain situations, the ratio of oligonucleotide- tethered nucleotide to a corresponding native nucleotide range from 1: 1 to 1 : 1000, more particularly 1 : 10, 1 :50, or 1 : 100. In some aspects, the sample is contacted with two or more oligonucleotide-tethered thymine, adenine, guanine, cytosine, or uracil nucleotides.

[0192] III. System

[0193] Method aspects of the present disclosure using compounds as described above may be implemented using a system comprising an extraction module and at least one container comprising at least one magnetic particle bound to at least one cell, such as a permeabilized cell, wherein the permeabilized cell comprises at least one oligonucleotide useful for directly tagging nucleic acids (sample nucleic acids or polynucleic acids) with specific oligonucleotide sequences. The oligonucleotides may be used as reagents in a nucleic acid synthesis reaction such that the nucleotide with an oligonucleotide tether may be incorporated into a nucleic acid formed from the synthesis reaction such as, for example, an extension reaction, amplification reaction, TdT reaction or the like. This allows for the incorporation of various types of functional sequences such as sequencing adapters, promoter sequences, barcodes, unique molecular identifiers, handle sequences, and the like into nucleic acids. The incorporation can either direct if the oligonucleotide of the oligonucleotide-tethered nucleotide provides a functional sequence, or indirect, such as by providing sequences that enable the addition of various functional sequences.

[0194] In some aspects, the extraction module may comprise a single magnet, but can alternatively comprise multiple magnets in order to provide a greater magnetic flux to capture the magnetic particles bound to a cell. The extraction module functions to extract at least one magnetic particle bound to a cell from a container or can be used to extract multiple magnetic particles bound to a cell or cells from an array of containers. The at least one magnetic particle bound to a cell can be removed by applying a removal force. The removal force can be applied by aspirating the contents out of a well. In some aspects, the removal force is a negative pressure. The removal force additionally or alternatively can be applied by pumping fluid through the array of wells, for example, by way of a perimeter channel, to provide a positive pressure that drives the at least one magnetic particle bound to a cell from the well.

[0195] FIG. 3 illustrates an extraction module 300 that functions to selectively remove one or more cells comprising a nucleic acid 310 bound to a magnetic particle 320 from a first liquid mixture 330. As depicted in FIG. 3, the extraction module 300 is configured to access at least one well 340 or an array of wells from a direction normal to the upper broad surface of the first liquid mixture 330. The extraction module 300 can include an outer layer 350 and an inner core comprising a magnet 360 to provide a magnetic flux and thus attract the magnetic particles 320. As depicted in FIG. 3, magnet 360 is inserted into the extraction module 300 through a first end 370. Magnet 360 attracts the magnetic particles 320 from liquid mixture 330 to the second end 380 opposite the first end 370 of the extraction module 300, allowing for the removal of magnetic particle 320 bound to one or more cells comprising a nucleic acid 310. Moreover, as illustrated in FIG. 3, magnet 360 may be removed from first well 340, and operatively positioned adjacent a second well 390. Thus, the magnetic particle 320 bound to one or more cells comprising a nucleic acid 310 can be introduced into a second well 390 comprising a second liquid mixture 395.

[0196] In some aspects, the container is further coupled to a magnet holder configured stabilize the position of the magnet or magnets of the extraction module. Additionally, the magnet can be positioned proximal to the container, or the magnet can be unfixed or fixed relative to any other element of the system. In one aspect, illustrated by FIG. 4, a container such as a well plate 400 is affixed to a magnet 410 is a rectangular prismshaped magnet affixed to a well plate 400 comprising at least one well 420 comprising at least one magnetic particle bound to at least one cell (not shown). The magnet 410 is affixed near a bottom end 430 of the at least one well 420 of the well plate 400 such that the magnetic particles bound to a cell are attracted toward the bottom of the well thereby allowing for the removal of supernatant used in cell processing. In another exemplary aspect, the magnet is configured to a microwell plate comprising 96 wells, wherein the microwell plate is comprised of from 1 to 96 wells corresponding to the microplate. Other variations such as SUPERMAG BEAD SEPARATION 96PCR (V&P Scientific Inc) and the like can be used.

[0197] In some aspects, the system may further be configured to a rack. A rack may hold the at least one container or more containers comprising at least one magnetic particle bound to at least one permeabilized cell and placed near magnet, such that when the magnetic field is applied to the container the bead-bound cells are gently drawn and retained on the container wall, which facilitates the removal of supernatant. Moreover, this aspect allows for the rack to be quickly removed from the magnet allowing for washing without removing the containers during the isolation process. Illustrated by FIG. 5 is a rack 500 configured for handling at least one container having a volume from greater than 0 mL to 50 ml. In some aspects, the container configured to the rack 500 depicted by FIG. 5 may include a main portion having an outer diameter, and a tapered end portion that tapers to a tip, for example, a microcentrifuge tube, such that the microcentrifuge tube can be placed through the openings 510 sized to accommodate the outer diameter of microcentrifuge tube. The containers may be pressed into openings 510, so that tapered portions of the containers press into openings 510 and constrain the containers within the rack. Moreover, FIG. 5 depicts a first support 520, a second support 530 and third support 540, wherein the first support 520 comprises an aperture 550 and the third end comprises an aperture 560 to be further configured to an elongated support.

[0198] Illustrated in FIG. 6, a magnet 600 operates in conjunction with the rack 610, wherein the magnet 600 has a rectangular shape having a first side 620 affixed to a first elongated support 630, a second side 640 affixed to second elongated support 650, and a bottom end 660 affixed to a base 670, and a top end 680 configured to the rack, wherein the magnets align with the containers held by the rack 670. As depicted by FIG. 6, the first elongated support 630 and second elongated support 650 is configured to fit through the aperture of the rack 610 and comprises a hollow opening 690 such that a support of the rack can be configured through the hollow opening 690; thus, can be easily removed from the magnet 600. Other variations such as the DynaMag™-Spin (Thermo Fisher), DynaMag™-2 (Thermo Fisher), DynaMag™-5 (Thermo Fisher), DynaMag™-15 (Thermo Fisher), DynaMag™-50 (Thermo Fisher), and the like can be used.

[0199] In some aspects, the system may be further configured for high throughput, single cell-based assays to enable extraction and analysis of 20 to at least 500,000 samples per day, such as 20-200 samples per day, or 200-1,000 samples per day, 500- 10,000 samples per day, 5,000-50,000 samples per day, 25,000-100,000 samples per day, 50,000-500,000 samples per day. FIG. 7 illustrates a system 700 for performing a high-throughput single cell-based assay. System 700 comprises a container 710 for holding pipette tips or aliquot of a sample, a container 720 comprising eight troughs 725(a)-(h), a first 96-well plate 730, a second 96-well plate 740, a third 96-well plate 750, a fourth 96-well plate 760, a fifth 96-well plate 770, an extraction module 780, and a 12-channel pipette 790. The wells in the 96-well plates may have a various volume, such as from greater than 0 pL to 5 mL. In some aspects, the extraction module comprises from 1 to 96 magnets. For a particular aspect, each magnet is rod-shaped having a diameter of from greater than 0 inches to 30 inches, a width of from greater than 0 inches to 40 inches, and a height of from greater than 0 inches to 25 inches. Each rod-shaped magnet is attached to a base.

[0200] The system may further include a controller storing instructions in non- transitory memory for the automated, semi-automated, or manual removal of magnetic particles bound to at least one cell. In some aspects, the system may further comprise one or more sensors for measuring a variety of physical properties, and one or more actuators. The system may further comprise a signaling means that converts measurements from the sensors and / or instructions generated by the controller into one or more signals sent to the extraction module and to at least one container. The controller may receive input data from one or more sensors, process the data. The input data may comprise instructions or code programmed therein corresponding to one or more routines, procedures, functions, methods, etc. The system may operate according to an open-loop system, closed-loop system, sequence control system, and / or batch system. The system may further comprise storage, one or more input devices, one or more output devices, one or more communication connections, and an operating system providing an operating environment for other software executing in the system, and coordinates activities of the components of the system. The communication connection enables communication over a communication mechanism to another computing entity. The communication mechanism conveys information such as computer-executable instructions, audio / video or other information, or other data. In some aspects, the communication mechanisms may include wired or wireless techniques implemented with an electrical, optical, RF, infrared, acoustic, or other carrier. The techniques herein can be described in the general context of computer-executable instructions, such as those included in program modules, being executed in a computing environment on a real or virtual processor. Generally, program modules include routines, programs, libraries, objects, classes, components, data structures, etc., that perform particular tasks or implement particular abstract data types. The functionality of the program modules may be combined or split between program modules as desired in various aspects. Computer-executable instructions for program modules may be executed within a local or distributed computing environment.

[0201] Any of the disclosed methods can be implemented as computer-executable instructions or a computer program product stored on one or more computer-readable storage media such as non-transitory computer-readable media. For example, one or more optical media discs such as DVD or CD, volatile memory components such as DRAM or SRAM, or non-volatile memory components such as hard drives and executed on a computer such as any commercially available computer, including smart phones or other mobile devices that include computing hardware. Computer-readable media does not include propagated signals. Any of the computer-executable instructions for implementing the disclosed methods as well as any data created and used during implementation of the disclosed aspects can be stored on one or more computer-readable media such as non-transitory computer-readable media. In some aspects, the computer-usable or computer-readable medium may be an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, device, or propagation medium. In particular disclosed aspects, the computer-readable medium may include an electrical connection having one or more wires, a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), an optical fiber, a portable compact disc read-only memory (CD- ROM), an optical storage device, a transmission media such as those supporting the Internet or an intranet, or a magnetic storage device. In some aspects, the computer- usable or computer-readable medium can be paper or another suitable medium upon which the program is printed, as the program can be electronically captured, via optical scanning of the paper or other medium, then compiled, interpreted, or otherwise processed in a suitable manner, if necessary, and then stored in a computer memory. In the context of this document, a computer-usable or computer-readable medium may be any medium that can contain, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device. The computer-usable medium may include a propagated data signal with the computer- usable program code embodied therewith, either in baseband or as part of a carrier wave. The computer usable program code may be transmitted using any appropriate medium, including but not limited to wireless, wireline, optical fiber cable, RF, and the like.

[0202] In some aspects, the computer-executable instructions can be part of a dedicated software application or a software application that is accessed or downloaded via a web browser or other software application such as a remote computing application. In particular disclosed aspects, the software can be executed on a single local computer such as in any suitable commercially available computer or in a network environment. For example, via the internet, a wide-area network, a local-area network, a client-server network such as a cloud computing network, or other such network using one or more network computers. The method may be implemented by hardware, software, firmware, middleware, microcode, hardware description languages, or any combination thereof. When implemented in software, firmware, middleware or microcode, the program code or code segments to perform the necessary tasks may be stored in a machine or computer readable medium. A code segment may represent a procedure, a function, a subprogram, a program, a routine, a subroutine, a module, program code, a software package, a class, or any combination of instructions, data structures, program statements, and the like.

[0203] With reference to FIG. 8, some aspects of the extraction module may comprise a plurality of magnetic rods such as the KingFisher™, 96 Deep-well head (Thermo Fisher) (not shown), which may be configured to a cover 800 having a first end comprising an opening 810 through which each magnetic rod can be inserted, a hollow inner core 820 such that a extraction module can be inserted, and a second end having a tapered closed end that forms a tip 830 to provide enhanced magnetic attraction and separation of the magnetic particles 840 bound to a cell 850 via a ligand 860 from the supernatant.

[0204] In some aspects, the system may comprise the device described in U.S. Patent 6,448,092 and / or apparatus described in U.S. Patent 6,447,729, which is hereby incorporated by reference in its entirety. In an exemplary aspect, the system is automated via the KingFisher™ Flex Purification System, wherein the extraction module is the KingFisher™, 96 Deep-well head (Thermo Fisher), the first, second, and third well plate is the KingFisher 96 KF plate (200pL) (Thermo Fisher), and the fourth and fifth plate is the Microtiter deep well 96, V-bottom (Thermo Fisher). In one exemplary aspect, the system is configured for use with Bindit software, wherein a method for preparing a single cell RNA sequencing library is automated by providing instructions to the controller of the system.

[0205] IV. Methods for Tagging a Nucleic Acid Within a Cell

[0206] Described herein are aspects of a method for binding at least one magnetic particle to a permeabilized cell and incorporating an oligonucleotide into a nucleic of the permeabilized cell for library preparation by tagmentation, thereby simplifying the library preparation process. Aspects of the method may include steps to fix and permeabilize cells such that nucleic acids and proteins remain intact and fixed within their cell of origin, enabling reagents such as polymerases, nucleotides, primers, and the like, to enter cells bound to a magnetic particle. Suitable reagents are used for, for example, reverse transcription, primer extension, ligation amplifications, and the like. Functionalized magnetic particles bound to a cell can be extracted via an extraction module for cell washing after such reaction, and such cells therefore can be prepared for further downstream applications, such as subsequent splitting and pooling.

[0207] In some aspects, the methods include:

[0208] (a) contacting a permeabilized cell comprising at least one cellular nucleic acid and a functionalized magnetic particle comprising at least one ligand, to produce a mixture comprising the permeabilized cell associated with the ligand of the functionalized magnetic particle;

[0209] (b) contacting the at least one cellular nucleic acid with a first oligonucleotide, at least one nucleotide, a first primer, and a first polymerase under conditions to produce a first tagged nucleic acid strand in the permeabilized cell; and

[0210] (c) applying a magnetic field to the mixture, thereby separating the permeabilized cell associated with the ligand of the functionalized magnetic particle and the first tagged nucleic acid strand from other components of the mixture; wherein steps (a) and (b) can occur in either order.

[0211] In some aspects, the magnetic bead is bound to at least one cell by adding the magnetic particles to a container comprising a cell concentration of from greater than 0 cells / pL to 1000 cells / pL, such as from 300 cells / pL to 700 cells / pL, from 400 cells / pL to 600 cells / pL, from 500 cells / pL to 600 cells / pL, from 515 cells / pL to 530 cells / pL. In some aspects, the cells are fixed by adding a fixation solution. The cells are incubated in the fixation solution for a suitable time, such as from greater than 0 to 20 minutes. Fixation agents may comprise formaldehyde, formalin, methanol, paraformaldehyde, methanol, acetic acid, and the like. Cells can be fixed by contacting the cells with 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, (v / v) or more, or any range in between, of formaldehyde or paraformaldehyde, or for example in phosphate buffered saline (PBS). In an exemplary aspect, the fixation solution comprises 20 pL of SUPERase-in and 125 pL of 16% formaldehyde.

[0212] Agents for permeabilizing cells include, but are not limited to, triton 100, saponin, Tween 20, and organic solvents such as methanol, acetone and the like. For example, cells may be treated with 0.01 to 10% (v / v) TritonX 100, and 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more formaldehyde.

[0213] After the cells are incubated, a quenching solution is added to the container and centrifuged. In an exemplary aspect, the quenching solution may comprise from greater than 0 pL to 500 pL of IX PBS (pH 7.4) and from greater than 0 pL to 1000 pL of Tris HC1 (750 mM, pH 7.5).

[0214] In some aspects, the fixed cells are cryo-stored and thawed by resuspending in a cell dilution buffer. In an exemplary aspect, the cell dilution buffer may comprise an aqueous solution; IX PBS (pH 7.4), BSA (50mg / mL), and SUPERase-In.

[0215] In some aspects, the permeabilized cell bound to a magnetic particle are split (distributed) into a plurality of different wells of a multi-well plate or tube, such that each individual cell is randomly distributed.

[0216] Each of the permeabilized cells bound to a magnetic particle via a ligand such as Concanavalin A may be subjected to a first extension reaction to generate a first extension product. Reverse transcriptase can be used for the first extension reaction to generate a first extension product that is cDNA. The nucleic acid in each of the permeabilized cells bound to a magnetic particle via a ligand such as Concanavalin A may be contacted with (1) a polymerase, such as reverse transcriptase, to analyze RNA sample nucleic acids / polynucleotides, and (2) a first primer, such as a reverse transcriptase extension primer that includes a first barcode. In some aspects, the first bar code is a first extension barcode or a first well-specific barcode that is unique to each of the different permeabilized cells bound to a magnetic particle via a ligand such as Concanavalin A, nucleotides, and at least one oligonucleotide-tethered ddNTP that includes a universal handle. The contacting step can be done under extension conditions. For whole transcriptome analysis, the first extension primer can include a sequence to enable amplification of mRNAs. As such, first extension primers can include a poly(T) to enable priming of mRNAs. Alternatively, the first extension primers can include a sequence to facilitate random priming of cellular RNAs such as hexamers. In some aspects, the poly(T) or random primer is provided at the 3’ end of the first extension primer. In some other aspects, a combination of primers with poly(T) and primers that include a sequence to facilitate random priming are used in the extension reaction.

[0217] In some aspects, the first extension primer can include a target-specific sequence. For target mRNA or DNA, the first extension primer can include a sequence that specifically hybridizes to the target sequence or target sequences of interest. An oligonucleotide-tethered binding agent can be used for analyzing proteins or cell markers that specifically binds to the protein, biomolecule, or cell marker of interest and includes an associated oligonucleotide sequence that identifies the binding agent is allowed to bind to the sample cells prior to fixation. The oligonucleotide-tethered binding agent functions in the same manner as a target-specific sequence used to analyze a target nucleic acid. Thus, the oligonucleotide-tethered binding agent can bind a protein and can be used translate data on protein expression into a format that can be analyzed using equipment and methodology used for sequencing of nucleic acids. Methods that use fluorophore-labeled antibodies can have issues with spectral fluorophore overlap. Additionally, methods with oligonucleotide-tethered binding agents can be performed without a need for using a fluorophore and equipment for analysis of fluorophores.

[0218] Multiplexing the combinatorial barcoding workflow allows the transcriptome and one or more target biomolecules to be processed and analyzed simultaneously. Moreover, cell populations can be contacted with one or more oligonucleotide-tethered binding agents. In some aspects, the cell populations may be contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more oligonucleotide-tethered binding agents that may include an oligonucleotide comprising a binding agent index and a 3’ poly(A) tail. The fixed and permeabilized cells are bound to a functionalized magnetic particle, which can be split via a magnet into the first compartments and processed the same way as cells are processed for whole transcriptome analysis.

[0219] In some aspects the first compartments are wells in a multi-well plate or tubes. The wells or tubes can be provided with a first extension primer and oligonucleotide- tethered ddNTPs. In some aspects, the wells in a multi-well plate or tubes can further include a polymerase, nucleotides, or both. In some aspects, the wells in a multi-well plate or tubes can further comprise pre-loaded first extension primers, and the like. The first extension results in the random incorporation of oligonucleotide-tethered ddNTPs into the first extension products, such as cDNA when the sample nucleic acids to be analyzed are RNA, thus generating random termination points of each first nucleic acid extension product. The “stop” point of each extension product advantageously can be used as an identifier to determine the progeny of each first extension product.

[0220] Each first nucleic acid extension product includes a first barcode (at its 5’ end) that is common to all of the nucleic acid extension products to each individual permeabilized cell bound to a magnetic particle via a ligand such as Concanavalin A and unique relative to the first barcodes in other compartments / first extension products generated in other compartments, and a second universal handle at the 3’ end that provides a 3’ OH that can be extended in a subsequent reaction. The permeabilized cells bound to a magnetic particle via a ligand such as Concanavalin A are washed with a first wash buffer, combined, or pooled, for example, into a single tube or well, and subsequently split into a plurality of second portions, for example, within individual wells in a multi-well plate, tubes, or the like. The individual cells are thus randomly distributed among individual second portions. In some aspects the first wash buffer may have a volume from greater than 0 pL to 200,000 pL. In some aspects, the first wash buffer may comprise from 0 pL to 50,000 pL of aqueous solution, from 0 pL to pL ATP (100 mM) from 0 pL to 50 pL of MgCl2, from 0 pL to 15,000 pL of SSIV Buffer, from 0 pL to 60,000 pL of PBS, from 0 pL to 1,000 pL of BSA, from 0 pL to 1,000 pL of digitonin, from 0 pL to 15,000 pL of Tris HC1 (50mM), from 0 pL to 500 pL of SUPERase-In. In some aspects, the second portions are contacted with splint oligonucleotide(s), a polymerase, and nucleotide(s). The splint oligonucleotides may comprise a sequence at the 3’ end that anneals to the second universal handle on the first nucleic acid extension product. The splint oligonucleotides may also contain a modification at the 3’ end, for example, a 3’ amino, a 3’ phosphate, a 3’ dideoxynucleotide, or the like, to prevent extension of the splint. The individual cells are thus washed again with a second wash buffer, combined / pooled, and split into a plurality of third portions, e.g., within individual wells in a multi-well plate, tubes, or the like. The individual cells are thus randomly distributed among individual third portions. In some aspects the second wash buffer may have a volume from greater than 0 pL to 200,000 pL. In some aspects, the second wash buffer may comprise from 0 pL to 50,000 pL of aqueous solution, from 0 pL to pL ATP (100 mM) from 0 pL to 50 pL of MgCl2, from 0 pL to 15,000 pL of SSIV Buffer, from 0 pL to 60,000 pL of PBS, from 0 pL to 1,000 pL of BSA, from 0 pL to 1,000 pL of di gitonin, from 0 pL to 15,000 pL of Tris HC1 (50mM), from 0 pL to 500 pL of SUPERase-In.

[0221] A template sequence for a third universal handle sequence may be provided at the 5’ end of the splint oligonucleotide. Between the sequence that hybridizes to the second universal handle and the third universal handle, the splint oligonucleotides include a template sequence for a second barcode that is unique to each second portion. As such, the first extension products within the second portions are further extended to generate second extension products that comprise, from the 5’ to 3’ direction, a first universal handle sequence, cDNA, dideoxynucleotide, second universal handle, second barcode and a third universal handle.

[0222] In some examples, a third barcode and fourth universal handle can be added to the nucleic acid samples of the sample. Specifically, second portions can be combined / pooled, and split into a plurality of third portions within the wells in a multiwell plate, tubes, or the like. The individual cells are thus randomly distributed among individual third portions. Extension reactions using a second set of splint oligonucleotides to add barcodes unique to each portion following pooling and splitting can be performed multiple times, each iteration resulting in the addition of another barcode unique to each portion (but common to all cells distributed to a particular portion) to the nucleic acids.

[0223] In some aspects, the method includes a step of removing, blocking, or digesting splint oligonucleotides, which can be after generating the second nucleic acid extension products and prior to contacting a third portion with a second extension primer. In some aspects, the splint oligonucleotides may comprise a binding moiety, and the method comprising contacting the second portions or combined second portions with a compound comprising a capture moiety that facilitates binding and removal of splint oligonucleotides comprising cognate binding moieties. In some aspects, the binding moiety and the cognate capture moiety are a binding pair as will be understood by a person of ordinary skill in the art, such as binding pairs selected from streptavidin and biotin, maltose and maltose binding protein, glutathione and glutathione S-transferase, chitin and chitin binding protein, or an aptamer and its antigen. In some aspects, the capture moiety is immobilized on a solid support. In some aspects, the solid support comprises a bead. In some aspects, the bead is a magnetic or paramagnetic bead.

[0224] In some aspects, OTDDNs can be incorporated into single cell or single nuclei workflows, such as whole transcriptome analysis, whole genome analysis, directed mRNA analysis, short RNA (for example, miRNAs), and the like, and combinations of these. A polymerase, such as DNA polymerases, RNA polymerases, reverse transcriptase, and telomerases, may be used to incorporate the oligonucleotide-tethered nucleotide into a nucleic acid synthesis product. For example, modified nucleoside triphosphates are recognized by DNA polymerases commonly used for primer extension and amplification protocols such as, for example, thermostable DNA polymerases including Taq polymerase, Vent polymerase, Pfx polymerase, Pwo polymerase, or Therminator polymerase. Alternatively, modified nucleoside triphosphates are accepted by, for example, mesophilic polymerases, such as MMLV reverse transcriptase, T7 DNA polymerase, and terminal deoxynucleotide transferase.

[0225] In some aspects, polymerases incorporate particular oligonucleotide-tethered nucleotides. In some aspects, the polymerases may be DNA and RNA polymerases. The DNA polymerase may be a DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase, or a template-independent DNA polymerase. The RNA polymerase may be a DNA-dependent RNA polymerase, or an RNA-dependent RNA polymerase, or a template-independent RNA polymerase. In some aspects, the polymerases may be wild-type, mutant isoforms, chimeric forms, and genetically engineered variants, such as exo-polymerases and other similar mutants. In some aspects, the polymerase is selected from an A family DNA polymerase, a B family DNA polymerase, an X family polymerase, a RT family polymerase, and variants and derivatives thereof.

[0226] In some aspects, the polymerase is a template-independent RNA polymerase, such as polyA polymerase (PAP) or polyU polymerase (PUP). In some aspects, the polymerase is a template-dependent RNA polymerase. In some examples, an RNA polymerase is a DNA-dependent RNA polymerase, such as T7 RNA polymerase, T3 RNA polymerase, T4 RNA polymerase, SP6 RNA polymerase and other RNA polymerases from T7-type bacteriophages. In other examples, an RNA polymerase is an RNA-dependent RNA polymerase, such as Q-beta replicase. These polymerases include wild-type, mutant isoforms, chimeric forms, and genetically engineered variants and mutants that tolerate modified nucleotides and may incorporate modified nucleotides into a nucleic acid. In some aspects, the A family DNA polymerase is a thermophilic or a mesophilic polymerase. In some aspects, the B family DNA polymerase is a thermophilic such as archeal or a mesophilic polymerase.

[0227] In some aspects, the DNA polymerase is an A family DNA polymerase chosen from a Pol I-type DNA polymerase, such as E. coli DNA polymerase, the Klenow fragment of E. coli DNA polymerase, polymerase from T. aquaticus (Taq DNA polymerase), T. thermophilus (Tth DNA polymerase), Bacillus stearothermophilus (Bst DNA polymerase), from bacteriophages such as T3 (T3 DNA polymerase) or T7 (T7 DNA polymerase), and variants and derivatives thereof. Variants and derivatives of A family DNA polymerases may be, for example, Thermo Sequenase™, CycleSeq™ (a combination of Taq DNA polymerase mutants capable of more efficiently incorporating modified nucleotides such as dideoxynucleotides), Stoeffel fragment (Truncated version of Taq DNA polymerase), Sequenase™ V2.0 (T7 DNA Polymerase mutant capable of more efficiently incorporating modified nucleotides such as dideoxynucleotides). In other aspects, the DNA polymerase is a B family DNA polymerase selected from Tli polymerase, Pfu polymerase, Pwo polymerase, KOD polymerase, Sac polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, phage Phi29 polymerase, and phage Bl 03 polymerase, a Type B polymerase from Pyrococcus and Thennococcus genera, such as the Pyrococcus strain GB-D (Deep Vent polymerase), P. furiosus (Pfu DNA polymerase), P. calidifontis (Pea DNA polymerase), P. aerophilum, T. kodakarensis (KOD DNA polymerases), T. gorgonarius (Tgo DNA polymerase), and Thermococcus sp.9°N-7 (9°N DNA polymerase); and variants and derivatives thereof. In some aspects, B type DNA polymerase is a modified Pyrococcus furiosus DNA polymerase. In some aspects the DNA polymerase is a chimeric Pyrococcus-like DNA polymerase fused with a dsDNA binding domain, for example, Phusion DNA polymerase, SuperFi DNA polymerase, Q5 DNA polymerase, Herculase II Fusion DNA polymerase, PfuUltra Fusion II HS DNA polymerase. In some aspects, DNA polymerase is Phusion exo- or other Pyrococcus-like DNA polymerase.

[0228] An exonuclease minus modification comprises modifications (D141A and E143A) or other respective modifications in an exonuclease domain to inhibit exonuclease activity. A suitable variant and derivative of a B family DNA polymerase may be, for example, Therminator polymerase (9°N™ DNA Polymerase variant with an enhanced ability to incorporate modified nucleotides such as dideoxynucleotides). In other aspects, the DNA polymerase is an X-type polymerase selected from Terminal deoxynucleotidyl Transferase (TdT), poly(A) polymerase, and poly(U) polymerase, and variants and derivatives thereof.

[0229] In other aspects, the polymerase is a reverse transcription polymerase selected from HIV reverse transcriptase, M-MLV reverse transcriptase, AMV reverse transcriptase, and variants and derivatives thereof.

[0230] In some instances, the polymerase is selected from Taq DNA polymerase, Vent® DNA polymerase, Deep VentTM DNA polymerase, Pfx DNA polymerase, Pwo polymerase, SuperScript™ IV (mutant MMLV RT), SuperScript™ II (mutant MMLV RT), SuperScript™ III (mutant MMLV RT), Maxima™ (mutant MMLV RT), RevertAid™ (mutant MMLV RT) reverse transcriptases, Thermo Sequenase™, Sequenase™ V2.0, CycleSeq™, Phusion exo-, Terminal deoxynucleotidyl Transferase (TdT), Maxima H (mutant MMLV RT), TherminatorTM polymerase, Q5 DNA polymerase, AccuTaq DNA polymerase, T7 DNA polymerase, T3 DNA polymerase, T4 DNA polymerase, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, DNA polymerase 1, KI enow fragment, Tth DNA polymerase, Phusion® DNA polymerase, SuperFi DNA polymerase, Platinum Taq DNA polymerase, Herculase II Fusion DNA polymerase, PfuUltra Fusion II HS DNA polymerase, Bst DNA polymerase large fragment, Stoeffel fragment, 9°N™ DNA polymerase, Pfu DNA polymerase, Tfl DNA polymerase, Phi29 polymerase, Tli DNA polymerase, eukaryotic DNA polymerase beta, telomerase, KOD HiFi DNA polymerase, KOD DNA polymerase, Q-beta replicase, AMV reverse transcriptase, M-MLV reverse transcriptase, Phi6 reverse transcriptase, and HIV-1 reverse transcriptase, polyA polymerase (PAP), polyU polymerase (PUP), and variants and derivatives thereof.

[0231] In some examples, the polymerase is selected from Taq DNA polymerase, SuperScript™ IV (mutant MMLV RT), Thermo Sequenase™, Sequenase™ V2.0, CycleSeq™, Phusion exo-, Maxima H (mutant MMLV RT), TherminatorTM polymerase, T7 DNA polymerase, T7 RNA polymerase, T3 RNA polymerase, Phusion (exo-) DNA polymerase, polyA polymerase (PAP), polyU polymerase (PUP), and variants and derivatives thereof.

[0232] In some aspects, the polymerase is capable of reading through a conjugation linker. Polymerases capable of reading through an unnatural linker provided upon incorporation of oligonucleotide-tethered nucleotide may be the same or different compared to the polymerases capable of incorporating an oligonucleotide-tethered nucleotide into a nucleic acid. DNA polymerases commonly used for primer extension and / or amplification protocols are used such as DNA-dependent DNA polymerases, RNA-dependent DNA polymerases, DNA-dependent RNA polymerases and RNA- dependent RNA polymerases.

[0233] Certain disclosed aspects concern preparing libraries of nucleic acids to be used in nucleic acid sequencing. The template nucleic acid sequence may be DNA or RNA. In some aspects, the template nucleic acid sequence is from double-stranded DNA and can be genomic DNA. Alternatively, the template nucleic acid sequence may be singlestranded DNA. In other aspects the template nucleic sequence may be RNA, mRNA, or miRNA. In some aspects, the template nucleic acids are nucleotide sequences provided by oligonucleotide-tethered binding agents.

[0234] In some aspects, library preparation for sequencing is performed by the following steps using an oligonucleotide-tethered nucleotide. First, a complementary primer that is annealed to the template nucleic acid strand is contacted with an oligonucleotide-tethered nucleotide and polymerase that are to be incorporated into a synthesized complementary nucleic acid strand. In some aspects, where a dideoxy version of the oligonucleotide-tethered nucleotide is used, the nucleic acid strand can be further extended by incorporating conventional nucleotides and oligonucleotide-tethered nucleotides. In some aspects, where a dideoxy version of the oligonucleotide-tethered nucleotide is used, further extension of the strand by the polymerase may be blocked by using a terminating group on the incorporated oligonucleotide-tethered nucleotide. In some aspects, the strand is then subjected to a primer extension-based reaction. In an exemplary aspect, the strand is subjected to PCR, including but not limited to, asymmetric PCR, indexing PCR, or a reverse transcription reaction and used in various sequencing methods. Some sequencing libraries may be prepared by using a tethered oligonucleotide that includes a universal handle sequence. A splint oligonucleotide is annealed to the universal handle sequence. The splint oligonucleotide may comprise sequencing adapters.

[0235] The amplification may, in some aspects, be achieved using polymerase chain reaction (PCR) or any isothermal DNA amplification method including, but not limited to, MDA, RCA, NASBA, LAMP, HD A, ICAN, NEAR and, EXPAR. The nucleic acid fragments may be quantified using a quantitative polymerase chain reaction, microarray, fluorometric or spectrophotometric analysis.

[0236] The technology described herein is not limited to any particular sequencing platform, and instead is generally applicable and platform independent. In some aspects, the technology is applicable to emulsion PCR-based methods, bead-based, and non-bead-based methods. For example, a nucleic acid fragment may be sequenced using any appropriate technique known in the art, such as Maxam-Gilbert, Sanger, pyrosequencing, sequencing-by-synthesis, sequencing-by-ligation, single-molecule realtime sequencing, mass spectrometry, massively parallel signature sequencing, polony sequencing, Illumina (Solexa) sequencing, semiconductor sequencing, DNA nanoball sequencing, Heliscope and single molecule sequencing.

[0237] Oligonucleotide-tethered dideoxynucleotides (OTDDN’s) can be used to vastly simplify and improve combinatorial barcoding methods. The quality of single cell / nuclei analysis protocols, such as protocols that are based upon combinatorial barcoding of biomolecules of interest, for example, cell surface molecules, proteins, nucleic acids including DNA, RNA, miRNA, and the like, are improved.

[0238] Aspects of the disclosed method for improving whole transcriptome analysis at the cellular level also are disclosed. The combinatorial barcoding workflows described herein can readily be adapted and multiplexed in order to analyze one or more specific nucleic acid and / or protein targets such as by using a first extension primer with a target-specific sequence. In some aspects, the first extension primer with a targetspecific sequence is target mRNA or DNA sequence of interest, or a nucleotide that is tethered to a biomolecule of interest, such as disclosed oligonucleotide-tethered binding agents. Similarly, the combinatorial barcoding workflows described herein can be adapted for whole genome analysis (WGA), by randomly tagging gDNA with an OTDDN as described elsewhere herein and following the combinatorial barcoding workflow provided herein.

[0239] In some aspects, the combinatorial barcoding methods provided herein may further comprise lysing the cells after generating one or more extension products. Additionally, the combinatorial barcoding methods provided herein can also include one or more amplification reactions following the addition of the last barcode. In some aspects, amplification primers can anneal to universal handles present at the 3’ and 5’ ends of the extension products. The amplification primers may include adapter sequences to enable the nucleic acid library to be processed and analyzed on a desired New Generation Sequencing platform. The combinatorial barcoding methods may also include one or more sample preparation steps including but not limited to purification of nucleic acids away from cellular debris following a lysis step, or the like.

[0240] After a desired number of barcodes have been added to the nucleic acid extension products within each cell, the final portions can be subjected to lysis conditions in order to lyse the cells and release the nucleic acid extension products, and the magnetic beads can be removed. Some aspects may comprise combining the second portions, splitting the combined second portions, splitting into third portions, and generating a third nucleotide extension product, wherein the combination of the first, second, and third barcode sequences (or complements thereof) within each third nucleotide extension product is unique to each nucleic acid extension product originating from a single cell. Thus, the combination of the first, second, and third barcode sequences may be almost entirely unique, although there may be a chance repeat of one or more barcodes.

[0241] The improved nucleic acid tagging and nucleic acid library preparation methods described herein can also be used to spatially resolve biomolecules of interest in tissue samples.

[0242] In some methods, a tissue sample can be contacted with an array of addressable primer that includes, in a 5’ to 3’ direction, an addressable barcode domain, a first barcode that encodes positional information on the array, and a hybridization domain that enables hybridization of the addressable primer to polynucleotides of the tissue sample. The addressable barcode can include information relating to the x-coordinate and the y-coordinate on the array; thus, a population of features or sites can be differentiated from each other according to relative location. Different molecules that are at different sites of an array can be differentiated from each other according to the locations of the sites in the array. An individual site of an array can include one or more molecules of a particular type. In some aspects, a site can include a single target nucleic acid molecule having a particular sequence or a site can include several nucleic acid molecules having the same sequence and / or complementary sequence, thereof. In some aspects, the addressable primers are covalently linked to a solid surface. The polynucleotides of the tissue sample can be RNA, such as mRNA, oligonucleotides of oligonucleotide-tethered binding agent’s bound to the tissue sample, or the like. Accordingly, the hybridization domain can facilitate hybridization to sequences of interest, or that enables hybridization to a specific target sequence, or that enables hybridization to oligonucleotides of oligonucleotide-tethered binding agents. The tissue sample can be contacted with the array of addressable primers to generate a first annealed complex in the presence of an oligonucleotide-tethered didoxynucleotide, nontethered nucleotides, and a polymerase. Extension of the first annealed complex incorporates the oligonucleotide-tethered nucleotide, such as an oligonucleotide- tethered dideoxynucleotide, into a first extension product. The first extension products comprising the incorporated OTDDN can be manipulated as described herein to produce a nucleic acid library suitable for NGS sequencing.

[0243] The addressable primers may be covalently linked to the different features on the array. The addressable primers can further include a cleavage domain for releasing at least a portion of the addressable primers from the feature on the array, wherein the released portion would comprise the positional barcode and the hybridization sequence. In some aspects, the addressable primers can include a sequence that facilitates cleavage by a restriction enzyme, upon extension of the addressable primer in the presence of the tissue nucleic acids.

[0244] The tissue section may be stained and photographed, before or after the first extension products are formed. Accordingly, the addressable barcode is correlated with a position within the tissue sample. In some aspects, the methods described herein can be performed on a plurality of consecutive tissue sections to generate a three- dimensional profile of the RNA and / or DNA of the biomolecule analyzed. As such, in some aspects, the barcode(s) in the addressable primers further comprise a z-coordinate.

[0245] V. Methods for Preparing a Nucleic Acid Library

[0246] Permeabilized cells bound to the magnetic particles allow for nucleic acids, proteins, and other biomolecules within the cell or nuclei to remain intact and fixed within their cell of origin, while also allowing reagents such as polymerases, nucleotides, primers, and the like to enter the cells or nuclei wherein they can function in synthesis reactions, such as reverse transcription, amplification, and the like. Additionally, single cell resolution can be achieved by combinatorial barcoding because the cells can be split, barcoded, washed, and pooled multiple times. In this way samples from a given cell can receive multiple barcodes based on the introduction of different barcodes through multiple rounds of splitting, barcoding, and pooling.

[0247] In some aspects, methods of preparing a nucleic acid library include: preparing a sample comprising a plurality of cells, wherein at least one cell of the plurality of cells comprises a cellular nucleic acid, and wherein the at least one cell is fixed and permeabilized; contacting the sample with a functionalized magnetic particle comprising at least one ligand, producing a mixture wherein the at least one fixed and permeabilized cell is bound to the at least one ligand; barcoding the cellular nucleic acid of the at least one fixed and permeabilized cell in the mixture; applying a magnetic field to the sample, thereby separating the mixture from the sample; lysing the plurality of cells in the separated mixture; and preparing a nucleic acid library from the lysed cells by providing at least one amplification primer to form an amplified nucleic acid.

[0248] FIG. 9 illustrates an exemplary aspect of a method for generating a nucleic acid library according to the present disclosure. For example, in Step 1, cells are input into a system for practicing the present disclosure, wherein the cell is bound to a functionalized magnetic particle. In some aspects, prior to inputting the cell, the cell bound to the functionalized magnetic particle is permeabilized. In some aspects, the cell bound to the functionalized magnetic particle is permeabilized after inputting the cell. In Step 2, the cells are split (distributed) into separate containers, wherein the containers comprise at least one nucleotide not tethered to an oligonucleotide, and at least one oligonucleotide-tethered dideoxynucleotide comprising a second universal handle sequence. In Step 3, an enzyme, such as reverse transcriptase is used to generate a complementary nucleic acid. In Step 4, the cells bound to the at least one functionalized magnetic particle are extracted from their respective containers and pooled together. In Step 5, the pooled cells bound to the magnetic particles are washed with a wash buffer. In Step 6, the cells bound to the functionalized magnetic particles are split into a container comprising a primer. In Step 7, the primers are annealed and thus extend the nucleic acid to form a tagged nucleic acid. In Step 8, the magnetic particles bound to the at least one permeabilized cell are extracted and pooled together. In Step 9, the magnetic particles bound to the at least one permeabilized cell are washed. In Step 10, the cells bound to the magnetic particles are split into different containers comprising Proteinase K. In step 11, the cells are lysed, and then washed with a lysis buffer. In Step 12, the DNA is extracted by transferring the lysis product out from each container to another container and thus separate the lysis product out from the magnetic particles. In Step 13, PCR amplifies specific regions of the tagged nucleic acid. In Step 14, the tagged nucleic acid is purified. In Step 15, the purified nucleic acid is pooled and prepared for output. In Step 16, the purified nucleic acid is then output for analysis.

[0249] In some aspects, reverse transcription may comprise annealing a first extension primer to one or more sample polynucleotides or sample nucleic acids; contacting the one or more sample polynucleotides or sample nucleic acids with a nucleic acid polymerase, at least one nucleotide, and an oligonucleotide-tethered nucleotide described herein to form a first extension product comprising the oligonucleotide- tethered nucleotide.

[0250] In some aspects, primer extension may comprise annealing a second primer which is at least partially complementary to the tethered oligonucleotide to form a second annealed complex; and contacting the second annealed complex with the nucleic acid polymerase to produce a nucleic acid molecule from the tethered oligonucleotide, thereby producing a library of polynucleotides comprising the tethered oligonucleotide sequence to at least one of its ends. In some aspects, a first extension primer and a second extension primer. The first extension primer may comprise a universal sequence and a second extension primer may comprise a universal sequence. In some aspects, first and second extension primers comprise different universal sequences. Thus, the first and second extension primers produce a library of polynucleotides comprising the universal sequence and tethered oligonucleotide sequence at its ends, after the incorporation of a tethered oligonucleotide.

[0251] In some aspects, the polynucleotides are fragmented prior to the step of annealing the first extension primer to the sample polynucleotides.

[0252] In some aspects, nucleic acids may be tagged with specific oligonucleotide sequences at predetermined or at random positions by using a combination of oligonucleotide-tethered nucleotides and corresponding unmodified native nucleotides. The frequency of random incorporation of oligonucleotide-tethered nucleotides may be controlled by modulating the molar ratio of oligonucleotide-tethered nucleotides relative to the corresponding native nucleotides.

[0253] In some aspects, where the first extension primer includes a universal sequence and a random sequence, the method can be used for whole genome or whole transcriptome sequencing. In some aspects, where the first primer comprises specific primers, the method is useful for targeted DNA / RNA sequencing.

[0254] The oligonucleotide-tethered nucleotides can also be used to facilitate addition of barcodes, unique molecular identifiers, handles and / or sequencing adapters, in a template dependent manner. Accordingly, some aspects concern adding nucleic acid sequences in a template-dependent manner to a sample polynucleotide. The method can include annealing a first primer to one or more sample polynucleotides or sample nucleic acids; and contacting the one or more sample polynucleotides or sample nucleic acids with a nucleic acid polymerase, at least one nucleotide, and an oligonucleotide- tethered dideoxynucleotide (OTDDN) described herein to form a first extension product comprising a copy of at least part of the one or more sample polynucleotides or sample nucleic acids with the OTDDN incorporated at the 3’ end. Desired information can be added to the 3’ end of the first extension product by contacting the extension product with a splint oligonucleotide comprising a sequence that hybridizes to the oligonucleotide portion of the OTDDN, and a template for a desired, additional sequence, in the presence of a polymerase and nucleotides, thereby producing a second extension product. Accordingly, desired additional sequences, such as barcodes, indexes, unique molecular identifiers, adapters (such as a sequencing adapter), a handle sequences, promoter sequences, or any combination thereof, or any other desired sequence, is added to the 3’ end of the tethered oligonucleotide of the first extension products.

[0255] In some aspects, the splint oligonucleotide can include a blocking group that prevents extension from the 3’ end of the splint. The splint can include a 3’ amino, a 3’ phosphate, a dideoxy, or other modification that prevents extension. In some examples the splint oligonucleotides can include a functional moiety, such as biotin / streptavidin or the like, to enable purification of the splint oligonucleotides.

[0256] The first primer that anneals to the sample nucleic acids or sample polynucleotides can include a hybridization sequence that enables annealing to the sample polynucleotides under extension conditions. In some aspects, the hybridization sequence can be a poly(T) tail that enables binding of the first extension primer to mRNA. In some aspects, the hybridization sequence can be a random sequence, for example a random hexamer, or the like, that enables non-selective binding of the first extension primer to sample polynucleotides. In some aspects, the hybridization sequence can be a target-specific sequence, such as a sequence that enables hybridization to a specific target nucleic acid sequence. In some aspects, the sample nucleic acids or sample polynucleotides are contacted with first primers that include more than one type of hybridization sequence. The hybridization sequence can comprise a mixture of first extension primers having a poly(T) hybridization sequence and first extension primers that have random hybridization sequences such as random hexamer sequences.

[0257] A single polymerase may be used to practice disclosed aspects. Alternatively, two different polymerases may be used, such as a first polymerase for incorporation of the oligonucleotide-tethered nucleotide and a second polymerase for primer extension / read-through. Polymerase reactions during which a polymerase incorporates an oligonucleotide-tethered nucleotide into a nucleic acid, reads-through an unnatural linker of incorporated oligonucleotide-tethered nucleotide, and / or extends a primer / oligonucleotide, may be performed under conditions suitable for the polymerase activity. In some aspects, a primer / template system, polymerase reaction buffer, incubation temperature and incubation time may be as typically recommended for a corresponding polymerase.

[0258] In another aspect TdT incorporates a single oligonucleotide-tethered dideoxynucleotide to the 3’ termini of single stranded or double stranded DNA / RNA in a template independent manner. Thus, adapters, such as for New Generation Sequencing and the like, can be added to sample nucleic acids. In some aspects, the oligonucleotide sequence of the OTDDN may include a first adapter sequence. Second adapters that are partially complementary to the first adapter sequences can be annealed and ligated to the template nucleic acids. The resulting library has adapters at both ends, the adapters having complementary and mismatched regions. The library may be further amplified.

[0259] The disclosed method may also be used for single cell or nuclei sequencing applications. The use of oligonucleotide-tethered dideoxynucleotides can improve single cell or nuclei sequencing methods that are not performed in traditional compartments such as wells or tubes by eliminating the need for pre-amplification of nucleic acids derived from single cells or nuclei. Furthermore, oligonucleotide-tethered dideoxynucleotides can be used to spatially resolve transcriptomic, genomic, and proteomic data in tissue samples.

[0260] As another example, the oligonucleotide-tethered nucleotides may be used in a method for tagging a nucleic acid with an oligonucleotide by annealing a primer to the nucleic acid, contacting the nucleic acid with an oligonucleotide-tethered nucleotide as described herein, and a polymerase, thereby producing the tagged nucleic acid.

[0261] In some examples, the nucleic acid is tagged with the oligonucleotide-tethered nucleotide during a gap-filing reaction or a nick translation. For example, doublestranded DNA is randomly labeled with a pre-designed specific sequence tag by incorporating an oligonucleotide-tethered dideoxynucleotide during the nick translation or gap fdling reaction. The frequency of oligonucleotide incorporation may be controlled by adjusting the DNA nicking rate. The resulting polynucleotide library will have pre-designed tags at the 3’ termini. As the average fragment length is controlled through nicking rate, tagging may be performed by complete substitution of a single native nucleotide with oligonucleotide-tethered nucleotide in nick translation or gap fdling reaction mixtures.

[0262] Template-independent DNA 3’ end labeling with any pre-designed sequence is achieved by incorporating an oligonucleotide-tethered dideoxynucleotides using terminal deoxynucleotidyl transferase (TdT). The oligonucleotide tethered to the dideoxynucleotide may have a blocked 3’ end (for example, bearing a 3’ phosphate or a 3’ amino modification, or a dideoxynucleotide), so that it is not extended. After a first round of labeling, a complementary strand is synthesized upon primer extension from the oligonucleotide conjugated to the dideoxynucleotide by any polymerase capable of reading through the conjugation linker. A second round of DNA end labeling is then performed as newly synthesized strands will have accessible 3’ ends.

[0263] Template-independent RNA 3’ end labeling with any pre-designed sequence may be achieved by incorporating oligonucleotide-tethered dideoxynucleotides using poly(A) or poly(U) polymerase.

[0264] The incorporated oligonucleotide may then serve as universal priming site for reverse transcription with the possibility to label 5’ ends via template switch activity of the reverse transcriptase. The resulting tagged cDNAs is converted to a sequencingready library through PCR, which in turn introduces platform-specific full-length adapters.

[0265] One-step primer extension, using a primer with a 5’ anchor corresponding to the full-length adapter, and termination by incorporation of oligonucleotide-tethered dideoxynucleotides bearing the second full-length platform-specific adapter sequence, enables generation of sequencing-ready single-stranded libraries, which may be subjected to sequencing without any other enzymatic manipulations. This provides advantage that less library preparation steps are needed. When RNA samples are used, such strategy provides a further advantage in that only synthesis of first strand cDNA is needed to achieve a sequencing-ready, single-stranded DNA comprising adapters at 5’ and 3’ ends.

[0266] The present methods may include performing at least one cleanup step. In some aspects, the cleanup step comprises removing removal of unincorporated dNTPs, primers, primer dimers, salts, and other contaminants. In an exemplary aspect, the cleanup step comprises the AMPure XP Bead-Clean Up protocol.

[0267] VI. Kits

[0268] Kits for implementing method aspects discussed herein and / or kits that contain compositions and / or systems discussed herein are also disclosed.

[0269] Certain disclosed kits are suitable for preparing a nucleic acid library for single cell analysis. In some aspects, the kit includes a functionalized magnetic particle comprising at least one ligand; and at least one reaction component for incorporating an oligonucleotide into a nucleic acid of a cell in a sample via a synthesis reaction, wherein the oligonucleotide is an oligonucleotide-tethered nucleotide having Formula 1, or a salt thereof:

[0270] Formula 1, wherein NB is a nucleobase; each of X and Q are independently chosen from, H, OH, N3, halo, alkyl, alkoxy, alkyl, alkenyl, alkynyl, acyl, cyano, amino, ester, and amido;

[0271] Z and Y are independently a bond, amino, amido, alkyl, alkenyl, alkynyl, thioether, sulfonyl, sulfonamido, ether, ketone, carbonyl, anhydride, ester, imido, urea, urethane, and combinations thereof; and CXN is chosen from alkylene, alkenylene, alkynylene, ketone, carbonate, ester, ether, anhydride, amido, amino, aminoalkylene, imino, imido, diazo, carbamate ester, phosphodiester, sulfide, disulfide, sulfonyl, sulfonamido, and a heterocyclic group containing from one to four N, O, S atom, or a combination thereof; and instructions for using the functionalized magnetic particles.

[0272] The kits may comprise a system comprising an extraction module and at least one container comprising at least one reagent. In some aspect, the container has volume from greater than 0 mL to 500 mL. In one exemplary aspect, the container is the Reagent Reservoir (Fisher Scientific).

[0273] In some aspects, the reagent is a binding buffer for magnetic bead activation, thereby reducing non-specific binding of other sample components, prevents the functionalized magnetic particle from disassociating from bound cells, and induces the association of cells to the functionalized magnetic particle. The binding buffer is provided in any suitable amount, such as from greater than 0 mL to approximately 500 mL. In some aspects, the binding buffer may comprise of from 0 to HEPES, KC1, MnCh, CaCl, and ultrapure ddFLO.

[0274] In some aspects, the at least one reagent is an oligonucleotide described herein. In some aspects, the container of the kit may further comprise a reaction component for incorporating the oligonucleotide into a nucleic acid via a synthesis reaction. In one example, the reaction component is a reverse transcription buffering agent comprising 5X SSIV, DTT, MgCh, dNTP, OTddATP, SUPERase-In, SuperScript, digitonin, water, or any combination thereof. In another example, the reaction component may comprise a ligation buffering agent comprising Tris HC1, MgCh, ATP, water, and / or any combination thereof. In another example, the reaction component may comprise a ligation buffering agent comprising 10X T4, DTT, nuclease-free water, T4 Ligase, Superase-In, digitonin 5%, and / or any combination thereof. In another example, the reaction component may comprise an extension buffering agent comprising 5X SSIV, dNTP, SuperScript IV RT, reverse transcription wash buffer, digitonin, and / or any combination thereof. In another example, the extension buffer may further comprise DTT. In some aspects, the kit may further comprise a washing buffer. In one example, the washing buffer may comprise a first barcode wash buffer comprising from 0 pL to 15,000 Tris HC1, digitonin, and / or from 0 uL to 1000 pL of bovine serum albumin. In another example, the washing buffer may comprise a second barcode wash buffer comprising from 0 pL to 60,000 pL of PBS, from 0 pL to 1000 pL of bovine serum albumin, from 0 pL to 1000 pL of digitonin, and / or any combination thereof. In another example, the washing buffer may comprise a reverse transcription washing buffer comprising from 0 pL to 15,000 pL of 5X SSIV, from 0 pL to 60,000 pL of aqueous solution, from 0 pL to 1000 pL bovine serum albumin, and / or any combination thereof. In another example, the washing buffer may comprise an extension wash buffer comprising from 0 pL to 60,000 pL of PBS and from 0 pL to 1,000 pL of bovine serum albumin.

[0275] In some aspects, the kit may further comprise a lysis buffer. In one example, the lysis buffer may comprise from 0 mL to 50 mL of aqueous solution, from 0 mL to 10 mL of NaCl (5 M) NaCl, from 0 mL to 5 mL of Tris (IM, pH 8.0), from 0 mL to 10 mL of EDTA (0.5 M, pH 8.0), and from 0 mL to 5 mL of SDS (10%), and / or any combination thereof.

[0276] The following examples are provided to illustrate certain features of particular disclosed aspects. A person of ordinary skill in the art will appreciate that the scope of the present disclosure is not limited to the particular features of these examples.

[0277] VII. Examples

[0278] The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.

[0279] Example 1

[0280] In this example, a nucleic acid library was generated for single cell analysis. The samples were first prepared such that they could be bound using Concanavalin A magnetic beads described below. Next, a nucleic acid library was prepared following the workflow illustrated in FIG. 10 with an oligonucleotide-tethered dideoxynucleotide (OTDDN). As depicted by FIG. 10, the S7-Me-BC-T30 primer can bind to the polyA tails of mRNA and allowing the reverse transcription. “S7” in FIG. 10, illustrates a sequence that serves as a handle for adding adapters during amplification. Moreover, adapters specific for Illumina sequencing methods, represented by P7, P5, Index7, and Index5 in FIG. 10, were added to allow Illumina sequencing. “Me” in FIG. 10 illustrates the mosaic end, which is the sequence used for sequencing on Illumina platforms. The cells from the sample were split, barcoded, and pooled multiple times such that the single cell receives a first barcode, represented by “RT Barcode” in FIG. 10, and receives a second barcode via ligation and extension represented by “Barcode 2” in FIG. 10. This process results in samples from a single cell having a unique set of multiple barcodes compared to other single cells that were in the same original sample. Thus, the split-pool workflow scales directly with the number of available barcodes, which allows for processing of cells in bulk, and is amenable to multiplexing.

[0281] Described hereafter is the detailed protocol used for the workflow illustrated in FIG. 10.

[0282] Sample Preparation: Cell fixation involves the following steps: (1) single cell suspension counting the cells on the Countess III such that no more than 20 million cells are used per fixation tube; centrifuging the cells at 500 g for 5 minutes at 4 °C; (2) aspirating and discarding the supernatant leaving only approximately 50 pL at the bottom of the tube; (3) on ice, bringing the total volume of the cell suspension to 1875 pL using IX PBS; (4) resuspending the cells by pipetting gently; (5) adding 20 pL of SUPERase-In, adding 125 pL of chilled 16% formaldehyde, pipetting mix gently on ice, and letting the cells in fixation solution incubate for 10 minutes; (6) at the end of the 10-minute incubation, immediately adding ImL of quenching solution comprising 482 pL of IX PBS (pH 7.4) and 618 pL of Tris HC1 (750 mM) (pH 7.5) and centrifuging the cells at 500 g for 5 minutes at 4 °C; (7) aspirating and discarding the supernatant leaving only approximately 50 pL at the bottom of the tube; (8) resuspending the cells in IX PBS at a cell concentration of approximately 1,000 cells / pL, which is verified by counting cells on the Countess III. Next, the fixed cells are cryo-stored by verifying that between 1 million and 10 million cells are stored per cryotube; adding DMSO to bring the concentration in the tube to 10%; transferring the cell suspension and DMSO to a cryotube; placing the cryotube(s) into a Freeze Cube; placing Freeze Cube into -80 °C overnight and potentially storing at -80 °C for up to six months. The cryo-stored fixed cells are thawed by: (1) removing cryotube from the - 80°C freezer; (2) placing the cryotube in a bead bath or water bath at 37 °C; (3) once the ice chunk in the tube has melted fully, but the tube and liquid is still cold, centrifuging at 500g for 5 minutes at 4 °C; (4) aspirating and discarding the supernatant leaving only approximately 50pL at the bottom of the tube; (5) resuspending the cells in 1.0 mL of cell dilution buffer comprising 5,300 pL of Nuclease-Free Water; 4,700 pL of IX PBS (pH 7.4); 100 pL of BSA (50mg / mL); 100 pL of SUPERase-In; allowing cells to rest for 5 minutes; (6) aspirating the total volume of the cell suspension and then attaching a 40 pm Flowmi strainer to the pipette tip and to a new tube to slowly pipette the cells through the strainer; (7) counting the cells on the Countess III and confirming that the cell concentration is approximately 521 cells / pL (5.21xl05cells / mL); (8) and placing on ice.

[0283] Binding Cells To Concanavalin A Coated Beads: First, Concanavalin A beads were prepared by: (1) transferring 180 pL of Concanavalin A bead slurry into 1.41 mL binding buffer comprising 200 pL of HEPES at pH of 7.9 (1 M); 100 pL of KCL (1 M); 10 pL of MnCh (1 M); 10 pL of CaCh (IM); and 9.86 mL (reach 10 mL) of ultrapure ddH2O; (2) mixing by pipetting and placing the tubes on a magnet stand to clear (30 s to 2 min); (3) withdrawing the liquid completely, and removing from the magnet stand; (4) adding 1.32 mL of binding buffer and mixing by pipetting; (5) placing on magnet stand to clear, withdrawing liquid, and resuspending in 180 pL of cell dilution buffer; (6) and holding the bead slurry at room temperature until cells are ready. Next, the 50 pL beads / mL cells were added to and rocked on a nutator for 5 minutes.

[0284] Reverse Transcription: (1) from a Master Barcode # 1 plate, aliquoting 1 pL of 5 pM Barcode # 1 from each individual well to a new plate; (2) sealing and placing the plate on ice; (3) ensuring the cells are fully resuspended, and adding 440 pL of cells diluted in cell dilution buffer at a cell concentration of 521 cells / pL to the RT Master Mix, which was prepared according to Table 1; (4) transferring the entire volume of RT Master Mix and Cells to one section of a 3-divided reagent reservoir; (5) placing the RT plate prepared at (1) on a colored plate holder; (7) adding 19 pL of RT Master Mix + Cells to each well in the plate; (8) sealing the plate with a foil seal and roller, and placing the plate on ice for 10 minutes; (9) placing the plate on a thermal cycler following protocol according to Table 2. Split-Pool and Ligation: (1) From a Master Barcode #2 Plate, aliquoting 1 pL of pre-annealed Barcode #2 to a new plate and then placed on ice; (2) removing the plate from thermocycler and the contents of the plate transferred to an Eppendorf tube;

[0285] (3) placing the Eppendorf tube on a tube magnet for 5 min to allow the solution to clear;

[0286] (4) aspirating and discarding the supernatant; (5) adding 2 mL of Barcode #1 Wash buffer comprising 9,800 pL of Tris HCI 50mM (pH 7.5), 100 pL of digitonin 5%, and 100 pL of BSA (50mg / mL); (6) placing on tube magnet for 5 min to allow solution to clear; (7) aspirating and discarding supernatant; (8) resuspending the cells and adding 2 mL of Barcode #1 Wash Buffer and placed on tube magnet for 5 minutes allowing solution to clear; (9) aspirating and discarding the supernatant; (10) resuspending the cells by adding 2 mL of Ligation Master Mix comprising 220 pL of 10X T4 Ligation Buffer, 22 pL of DTT (0.1 M), 1318 pL of Nuclease-Free Water, 110 pL of T4 Ligase, 110 pL of Superase-In, and 22 pL of digitonin (5%); (11) transferring the entire volume of Ligation Master Mix, cells, and beads to one section of a 3-divided reagent reservoir; placing the Barcode #2 plate prepared in (1) on a colored plate holder and adding 19 pL of Ligation Master Mix and cells to each well of the plate; (12) sealing plate with a foil seal and roller and placed on thermal cycle for incubation following protocol in Table 3; (13) remove plate for thermal cycler and adding 10 pL of 150 mM EDTA to each well of the plate; (14) pipetting the contents of each well into a precooled 2 mL Eppendorf tube and placing on tube magnet for 5 minutes allowing solution to clear; (15) aspirating and discarding the supernatant; (16) resuspending cells by adding 2.0 mL of Barcode #2 Wash Buffer comprising 13,000 pL of IX PBS (pH 7.4) and 130 pL of BSA (50 mg / mL) to the tube and placing on tube magnet for 5 minutes allowing solution to clear; (17) aspirating and discarding the supernatant; (18) resuspending cells by adding 2 mL of Barcode #2 Wash Buffer to the tube and placing on tube magnet for 5 min to allow solution to clear; (19) aspirating and discarding the supernatant; (20) resuspending the cells by adding 2 mL of Barcode #2 Wash Buffer and placing on tube magnet for 5 min to allow for solution to clear; (21) resuspending the cells by adding 100 pL of Barcode #2 Wash Buffer.

[0287] Split-Pool and Lysis: (1) aliquoting 10 pL from the fully suspended cells and counting on the Countess 3 FL (ReadyCount Green / Red Viability Stain) the cell number by adding green and red counts (total count will count magnetic beads); (2) ensuring a cell input of < 44,250 cells and the cell concentration should be approximately 5.07xl05cells / mL; (3) aliquoting 2 pL of 20 mg / mL of Proteinase K to each individual well of a new plate and placing on a colored plate holder; (4) adding 25 pL of Barcode #2 Wash Buffer and cells to each well of plate and sealing plate with foil seal and roller; (5) placing plate on vortex for 3 seconds then centrifuging at 1000 X g for 2 seconds; (6) placing the plate on thermal cycle and incubating according protocol in Table 4; (7) removing the plate from the thermal cycler (Lysis plate can be stored overnight in -80°C freezer for up to 1 month. Ampure Purification: (1) Removing AMPure XP beads from refrigerator and incubating at room temperature for 30 minutes and then placing beads on vortex on high for 1 minute; (2) allowing plate to thaw to room temperature (if plate was stored in - 80°C freezer); (3) prepare Indexing Plate by aliquoting 26 pL of Library Amplification Mix comprising 2,854.5 2 pL of Collibri Library Amp Master Mix and 5.5 pL of Phusion exo-polymerase to a 96-well plate; (4) aliquoting 1 pL of 50 pM Illumina Barcodes from each individual well to a new plate, sealing, and placing on ice; (5) centrifuging plate at 1000 X g for 20 seconds; (6) placing the plate on a 96-well plate magnet for 2 minutes or until solution clears; (7) transferring lysis product from each individual well to a new 96 well plate to separate out Concanavalin A beads and placing on vortex set at high for 30 seconds; (8) aliquoting 5,000 pL of AMPure XP Beads to three sections of a 3 -divided reagent reservoir; (9) changing pipette tips each time, add 46.8 pL of well resuspend AMPure XP Beads to each well of the plate containing lysis product, and sealing with foil seal and roller; (10) placing plate on a ThermoMixer with a 96-well plate block set to 25°C and 2000 RPM for 30 seconds; (11) briefly centrifuging plate and incubating at room temperature for 5 minutes; (12) placing plate on a 96-well plate magnet for 2 minutes or until solution clears; (13) aspirating and discarding supernatant; (14) washing beads by adding 220 pL of 80% ethanol comprising 10 mL of nuclease-free water and 40 mL of pure ethyl alcohol and incubating for 30 seconds; (15) aspirating and discarding ethanol; (16) repeat (14)-(15) two times; (17) centrifuging plate at 1000 g for 2 seconds; (18) placing plate on magnet and aspirating the remaining ethanol using a P20 pipette; (19) air drying beads for 2 minutes without over drying; (20) removing plate from magnet and adding 24 pL of Elution Buffer EB (Qiagen) to each well and sealing plate with foil seal and roller; (21) placing plate on a ThermoMixer with a 96-well plate block set to 37°C and 2000 RPM for 30 seconds; (22) briefly centrifuging plate; (23) placing the plate on a 96-well plate magnet for 2 minutes or until solution clears; (24) removing foil seal from indexing plate of “(3)” and transferring 23 pL of eluted product from each well into each matching well on the indexing plate and pipetting mix gently 5 times; (25) sealing plate with foil seal and roller and centrifuging plate at 1000 X g for 2 seconds; (26) placing plate on thermal cycler and incubating according to Table 5. cDNA Amplification: (1) Centrifuging plate at 1000 X g for 20 seconds; (2) transferring PCT product from each well to a 5.0 mL tube; (3) transferring 1080 pL of PCR product to four 2.0 mL tubes (4 tubes with 1080 pL); (4) vortex AMPure XP beads on high for 30 seconds; (5) adding 594 pL of beads to each tube, closing cap, and placing on vortex briefly; (6) incubate at room temperature for 5 minutes; (7) while incubating, preparing 15 mL of fresh 80% ethanol comprising 3 mL of nuclease-free water to 12 mL of pure ethyl alcohol; (8) briefly centrifuging each tube; (9) placing each tube on a 1.5-2.0 mL tube magnet for 2 minutes or until the solution clears; (10) while on the magnet, vortex AMPure XP beads on high for 30 seconds and preparing 4 new 2 mL tubes containing 270 pL of AMPure XP beads; (11) for each tube, transferring entire supernatant to the new tube containing 270 L of AMPure XP beads (should be a total of 8 tubes — four tubes with only beads inside, on the magnet; and four tubes with approximately 1,944 pL of supernatant and AMPure XP beads); (12) incubating at room temperature for 5 minutes; (13) placing each tube on a 1.5-2.0 mL tube magnet for 2 minutes or until the solution clears; (14) aspirating and discarding the supernatant; (15) washing beads by adding 2000 pL of 80% ethanol and incubating for 30 seconds; (16) aspirating and discarding the ethanol; (17) centrifuging tubes briefly; (18) returning tubes to the magnet and aspirating the remaining ethanol using a P20 pipette; (19) air drying the beads for 3 minutes without over drying; (20) removing the tubes from the magnet and adding 50 pL of Elution Buffer EB (Qiagen) to each tube, closing cap, and placing on vortex briefly; (21) briefly centrifuging tubes; (22) placing tubes on a ThermoMixer with a 24-well block for 1.5-2.0 mL tube set to 37°C and 300 RPM for 10 minutes and briefly centrifuging tubes; (23) placing each tube on a 1.5-2.0 mL tube magnet for 2 minutes or until the solution clears; (24) transferring the eluted libraries to a single 1.5 mL tube (total of approximately 200 pL of eluted libraries); (25) PCR products can be stored in -20°C freezer for up to 6 months

[0288] Illumina Sequencing: Tapestation analysis is performed by: (1) performing a 1 : 10 dilution of each library with nuclease-free water; (2) analyzing each diluted library using Agilent Tapestation High Sensitivity D5000 ScreenTape and reagents; (3) traces are shown in FIGS. 11 and 12. Next, Quantification is done by performing a 1 :20,000 dilution of each library by: (1) diluting 1 pL of stock library into 99 pL of nuclease- free water; (2) vortexing and centrifuging briefly; (3) diluting 1 pL of the 1: 100 library into 199 pL of nuclease-free water; (4) vortexing and centrifuging briefly; (5) analyzing using the Collibri Library Quantification Kit, or KAPA Library Quantification Kit, Universal (for Illumina Platforms). Next, sequencing was performed by using the following Run configuration when setting up a sequence run: 130 / 10 / 10 / 10 for DesignO; ensuring that libraries are sequenced with at least a 20% phiX spike. Example 2

[0289] In this example, a nucleic acid library was generated for single cell analysis. The samples were first prepared such that they could be bound using Concanavalin A magnetic beads. Next, a nucleic acid library was prepared following the workflow illustrated in FIG. 13 with an oligonucleotide-tethered dideoxynucleotide (OTDDN). As depicted by FIG. 13, the S7-Me-BC-T30 primer can bind to the polyA tails of mRNA and allow reverse transcription. “S7” in FIG. 13, illustrates a sequence that serves as a handle for adding adapters during amplification. Moreover, adapters specific for Illumina sequencing methods, represented by P7, P5, Index7, and Index5 in FIG. 13, were added to allow Illumina sequencing. “Me” in FIG .13 illustrates the mosaic end, which is the sequence used for sequencing on Illumina platforms. The cells from the sample were split, barcoded, and pooled multiple times such that the single cell can receive a first barcode, represented by “RT Barcode” in FIG. 13, and a second barcode was added via extension (no ligation), which is represented by “Barcode 2” in FIG. 13. This process results in samples from a single cell having a unique set of multiple barcodes compared to other single cells that were in the same original sample. Thus, the split-pool workflow scales directly with the number of available barcodes, allows for processing of cells in bulk, and is amenable to multiplexing. Described hereafter is the detailed protocol used for the workflow illustrated in FIG. 13.

[0290] Sample Preparation'. Cell fixation involves the following steps: (1) single cell suspension counting the cells on the Countess III such that no more than 20 million cells are used per fixation tube; centrifuging the cells at 500 g for 5 minutes at 4 °C; (2) aspirating and discarding the supernatant leaving only approximately 50 pL at the bottom of the tube; (3) bringing the total volume of the cell suspension to 1875 pL using IX PBS; (4) resuspending the cells by pipetting gently; (5) adding 20 pL of SUPERase-In, adding 125 pL of chilled 16% formaldehyde, pipetting mix gently on ice, and incubating cells in fixation solution for 10 minutes; (6) at the end of the 10- minute incubation, immediately adding ImL of quenching solution comprising 482 pL of IX PBS (pH 7.4) and 618 pL of Tris HC1 (750 mM) (pH 7.5) and centrifuging the cells at 500 g for 5 minutes at 4°C; (7) aspirating and discarding the supernatant leaving only approximately 50 pL at the bottom of the tube; (8) resuspending the cells in IX PBS at a cell concentration of approximately 1,000 cells / pL, which is verified by counting cells on the Countess III. Next, the fixed cells are cryo-stored by verifying that between 1 million and 10 million cells are stored per cryotube; adding DMSO to bring the concentration in the tube to 10%; transferring the cell suspension and DMSO to a cryotube; placing the cryotube(s) into a Freeze Cube; placing Freeze Cube into -80 °C overnight and potentially storing at -80 °C for up to six months. The cryo-stored fixed cells are thawed by: (1) removing cryotube from the -80°C freezer; (2) placing the cryotube in a bead bath or water bath at 37°C; (3) once the ice chunk in the tube has melted fully, but the tube and liquid is still cold, centrifuging at 500g for 5 minutes at 4 °C; (4) aspirating and discarding the supernatant leaving only approximately 50pL at the bottom of the tube; (5) resuspending the cells in l.OmL of cell dilution buffer comprising 5,300 pL of Nuclease-Free Water; 4,700 pL oflX PBS (pH 7.4); 100 pL of BSA (50mg / mL); 100 pL of SUPERase-In; allowing cells to rest for 5 minutes; (6) aspirating the total volume of the cell suspension and then attaching a 40 pm Flowmi strainer to the pipette tip and slowly pipetting the cells through the strainer; (7) counting the cells on the Countess III and confirming that the cell concentration is approximately 521 cells / pL (5.21x105 cells / mL); (8) and placing on ice.

[0291] Binding Cells To Concanavalin A beads. Concanavalin A beads were prepared by: (1) transferring 180 pL of Concanavalin A bead slurry into 1.41 mL binding buffer comprising 200 pL of HEPES at pH of 7.9 (1 M); 100 pL of KCL (1 M); 10 pL of MnCh (1 M); 10 pL of CaCh (IM); and 9.86 mL (reach 10 mL) of ultrapure ddHjO;

[0292] (2) mixing by pipetting and placing the tubes on a magnet stand to clear (30 s to 2 min);

[0293] (3) withdrawing the liquid completely, and removing from the magnet stand; (4) adding 1.32 mL of binding buffer and mixing by pipetting; (5) placing on magnet stand to clear, withdrawing liquid, and resuspending in 180 pL of cell dilution buffer; (6) and holding the bead slurry at room temperature until cells are ready. Next, the 50 pL beads / mL cells were added to conical vial and rocked on nutator for 5 minutes.

[0294] Reverse Transcription: (1) from a Master Barcode # 1 plate, aliquoting IpL of 5 pM Barcode # 1 from each individual well to a new plate; (2) sealing and placing the plate on ice; (3) ensuring the cells are fully resuspended, adding 440 pL of cells diluted in cell dilution buffer at a cell concentration of 521 cells / pL to the RT Master Mix, which was prepared according to Table 1 (see Example 1); (4) transferring the entire volume of RT Master Mix and Cells to one section of a 3-divided reagent reservoir; (5) placing the RT plate prepared at (1) on a colored plate holder; (7) adding 19 pL of RT Master Mix and cells to each well in the plate; (8) sealing the plate with a foil seal and roller, and placing the plate on ice for 10 minutes; (9) placing the plate on a thermal cycler following the protocol according to Table 6; (10) After incubation, pipetting contents of each well into a precooled 2 mL Eppendorf tube and placing on tube magnet for 5 minutes and allowing solution to clear; (11) aspirating and discarding the supernatant; (12) resuspending the cells and beads in 2 mL of RT Wash Buffer comprising 10,400 pL of 5X SSIV Buffer, 40,560 pL of Nuclease-Free Water, and 520 pL of BSA (50 mg / mL); (13) placing on tube magnet for 5 minutes and allowing solution to clear; (14) aspirating and discarding the supernatant; (15) resuspending the cells in and beads in 2 mL Extension Master Mix comprising 100 pL of 5X SSIV Buffer 100 pL dNTP Mix (lOmM), 100 pL of SuperScript IV RT (200 U / pL), 1480 pL of RT Wash Buffer, and 20 pL of digitonin.

[0295] Split-Pool and Extend Barcode: (1) adding 19 pL of Ext Master Mix and cells to the well of each plate; (2) sealing plate with a foil seal and roller and placing on ice for 15 minutes; (3) incubating plate according to Table 7; (4) removing plate from thermal cycler and pipetting contents of each well into a precooled 2 mL Eppendorf tube; (5) placing on tube magnet for 5 minutes and allowing solution to clear; (6) aspirating and discarding the supernatant; (7) resuspending the cells in 2 mL Extension Wash Buffer comprising 53,000 pL of IX PBS (pH 7.4) and 530 pL of BSA (50 mg / mL); (8) placing on tube magnet for 5 minutes and allowing solution to clear; (9) aspirating and discarding the supernatant; (10) resuspending the cells and beads in 100 pL Extension Wash Buffer; (11) aliquoting 10 pL from the fully resuspended cells and counting on the Countess 3 FL a cell input of greater than or equal to 44,250 cells (cell concentration should be approximately 5.07xl03cells / mL); (12) adding 25 pL of Extension Buffer and cells to well of plate comprising 2 pL of 20 mg / mL Proteinase K; (13) sealing and vortexing the plate for 3 seconds then centrifuging at 1000 X g for seconds; (14) placing on incubator following protocol in Table 8. Ampure Purification: (1) removing AMPure beads from refrigerator and incubating at room temperature for 30 minutes and then placing beads on vortex on high for 1 minute; (2) allowing plate to thaw to room temperature (if plate was stored in -80 °C freezer); (3) prepare Indexing Plate by aliquoting 26 pL of Library Amplification Mix comprising 2,854.5 2 pL of Collibri Library Amp Master Mix and 5.5 pL of Phusion exo-polymerase to a 96-well plate; (4) aliquoting 1 pL of 50 pM Illumina Barcodes from each individual well to a new plate, sealing, and placing on ice; (5) centrifuging plate at 1000 X g for 20 seconds; (6) placing the plate on a 96-well plate magnet for 2 minutes or until solution clears; (7) transferring lysis product from each individual well to a new 96 well plate to separate out Concanavalin A beads and placing on vortex set at high for 30 seconds; (8) aliquoting 5,000 pL of AMPure XP Beads to three sections of a 3 -divided reagent reservoir; (9) changing pipette tips each time, adding 46.8 pL of well resuspend AMPure XP Beads to each well of the plate containing lysis product, and sealing with foil seal and roller; (10) placing plate on a ThermoMixer with a 96-well plate block set to 25°C and 2000 RPM for 30 seconds; (11) briefly centrifuging plate and incubating at room temperature for 5 minutes; (12) placing plate on a 96-well plate magnet for 2 minutes or until solution clears; (13) aspirating and discarding supernatant; (14) washing beads by adding 220 pL of 80% ethanol comprising 10 mL of water and 40 mL of pure ethyl alcohol and incubating for 30 seconds; (15) aspirating and discarding ethanol; (16) repeating (14)-(15) two times; (17) centrifuging plate at 1000 g for 2 seconds; (18) placing plate on magnet and aspirating the remaining ethanol using a P20 pipette; (19) air drying beads for 2 minutes without over drying; (20) removing plate from magnet and adding 24 pL of Elution Buffer EB (Qiagen) to each well and sealing plate with foil seal and roller; (21) placing plate on a ThermoMixer with a 96-well plate block set to 37°C and 2000 RPM for 30 seconds; (22) briefly centrifuging plate; (23) placing the plate on a 96-well plate magnet for 2 minutes or until solution clears; (24) removing foil seal from indexing plate of “(3)” and transferring 23 pL of eluted product from each well into each matching well on the indexing plate and pipetting mix gently 5 times; (25) sealing plate with foil seal and roller and centrifuging plate at 1000 X g for 2 seconds; (26) placing plate on thermal cycler and incubating according to Table 9. cDNA Amplification: (1) centrifuging plate at 1000 X g for 20 seconds; (2) transferring PCT product from each well to a 5.0 mL tube; (3) transferring 1080 pL of PCR product to four 2.0 mL tubes (4 tubes with 1080 pL); (4) vortexing AMPure XP beads on high for 30 seconds; (5) adding 594 pL of beads to each tube, closing cap, and placing on vortex briefly; (6) incubating at room temperature for 5 minutes; (7) while incubating, preparing 15 mL of fresh 80% ethanol comprising 3 mL of water to 12 mL of pure ethyl alcohol; (8) briefly centrifuging each tube; (9) placing each tube on a 1 .5- 2.0 mL tube magnet for 2 minutes or until the solution clears; (10) while on the magnet, vortexing AMPure XP beads on high for 30 seconds and preparing 4 new 2 mL tubes containing 270 pL of AMPure XP beads; (11) for each tube, transferring entire supernatant to a new tube containing 270 pL of AMPure XP beads (should be a total of 8 tubes — four tubes with only beads inside, on the magnet; and four tubes with approximately 1,944 pL of supernatant and AMPure XP beads); (12) incubating at room temperature for 5 minutes; (13) placing each tube on a 1.5-2.0 mL tube magnet for 2 minutes or until the solution clears; (14) aspirating and discarding the supernatant;

[0296] (15) washing beads by adding 2000 pL of 80% ethanol and incubating for 30 seconds;

[0297] (16) aspirating and discarding the ethanol; (17) centrifuging tubes briefly; (18) returning tubes to the magnet and aspirating the remaining ethanol using a P20 pipette; (19) air drying the beads for 3 minutes without over drying; (20) removing the tubes from the magnet and adding 50 pL of Elution Buffer EB (Qiagen) to each tube, closing cap, and vortexing briefly; (21) briefly centrifuging tubes; (22) placing tubes on a ThermoMixer with a 24-well block for 1.5-2.0 mL tube set to 37°C and 300 RPM for 10 minutes and briefly centrifuging tubes; (23) placing each tube on a 1.5-2.0 mL tube magnet for 2 minutes or until the solution clears; (24) transferring the eluted libraries to a single 1.5 mL tube (total of approximately 200 pL of eluted libraries); (25) storing PCR products in a -20 °C freezer for up to 6 months.

[0298] Illumina Sequencing: quality control of sample was evaluated using the automated electrophoresis device 4200 TapeStation System (Agilent) by: (1) diluting a 1 : 10 dilution of each library with water; (2) analyzing each diluted library using Agilent Tapestation High Sensitivity D5000 ScreenTape and comparing to traces as shown in FIGS. 11 and 12 to analyze suitable size, quantity, and integrity of sample. Next, Quantification is done by performing a 1:20,000 dilution of each library by: (1) diluting 1 pL of stock library into 99 pL of nuclease-free water; (2) vortexing and centrifuging briefly; (3) diluting 1 pL of the 1 : 100 library into 199 pL of nuclease-free water; (4) vortexing and centrifuging briefly; and (5) analyzing using the Collibri Library Quantification Kit, or KAPA Library Quantification Kit, Universal (for Illumina Platforms). Next, sequencing was performed using the following Run configuration when setting up a sequence run: 130 / 10 / 10 / 10 for DesignO; ensuring that libraries are sequenced with at least a 20% phiX spike. Example 3

[0299] In this example, the King KingFisher™ Flex Purification System, KingFisher with a 96 Deep-well Head was used to wash fixed cells bound to the magnetic particles.

[0300] Sample Preparation: (1) suspending no more than 10 million cells per fixation tube on the Countess; (2) centrifuging the cells at 300g for 10 minutes at 4 °C and resuspending in PBS and 1% FBS to wash; centrifuging the cells at 300 X g for 10 minutes at 4°C; (3) aspirating and discarding the supernatant leaving only approximately 50pL at the bottom of the tube; (4) on ice, bringing the total volume of the cell suspension to 1440pL using IX PBS for every 5 million cells; (5) resuspending the cells by pipetting; (6) to the tube, adding SUPERase-In at volume according to Table 10; (7) filtering through 40 uM centrifuge top filter and adding BSA at a volume from Table 10; (8) adding chilled 16% formaldehyde at a volume from Table 10 to the tube; (9) and pipetting to mix gently on ice and incubating the cells in fixation solution for 10 minutes; (10) adding 10,000 pL of IX PBS, pH 7.4 and 100 pL of SUPERase-In 20 U / pL, and adding 6 mL after 10 minutes to fill 15 ml conical; (11) centrifuging the cells at 600 X g for 10 minutes at 4 °C and aspirating and discarding the supernatant leaving only approximately 50pL at the bottom of the tube; (12) resuspending the cells in 10 ml PBS and RI and centrifuging the cells at 600g for 10 minutes at 4 °C; (13) aspirating and discarding the supernatant leaving only approximately 50 pL at the bottom of the tube and resuspending the cells in cell dilution buffer comprising 5,300 pL water, 4,700 of IX PBS pL, 100 pL of BSA (50 mg / mL), and 100 pL of SUPERase-In at a concentration of 1 million cells / ml.

[0301]

[0302] Preparation of Concanavalin A: (1) transferring 180 pL Concanavalin A head slurry into 1.41 mL binding buffer, mixing by pipetting; (2) placing the tubes on a magnet stand to clear (30 s to 2 min); withdrawing the liquid completely, removing from the magnet stand; (3) adding 1.32 mL of binding buffer comprising 200 pL of IM HEPES (pH 7.9), 100 pL of IM KC1, 10 pL of IM MnCl2, 10 pL of IM CaCl2, and ultrapure ddH2O; (4) placing on magnet stand to clear, withdrawing liquid, and resuspending in 180 pL cell dilution buffer; (5) holding the bead slurry at RT until cells are ready; (6) adding 50 pL beads / mL cells to conical vial and rocking on nutator for 5 minutes.

[0303] Preparation of KingPisher Plates: (1) loading 200 pL of wash buffer comprising 49,000 pL of IX PBS (pH 7.4), 500 pL of BSA (50 mg / mL), and 500 pL of 5% digitonin per well in KingFisher DW Plate and placing the tip comb in plate to prime plastic; (2) loading 250 pL of wash buffer comprising 49,000 pL of IX PBS (pH 7.4), 500 pL of BSA (50 mg / mL), and 500 pL of 5% digitonin per well in KingFisher DW plate in wash plate; (3) loading 100 pL of wash buffer comprising 49,000 pL of IX PBS (pH 7.4), 500 pL of BSA (50 mg / mL), and 500 pL of 5% digitonin in KingFisher 200 pL plate for cell drop; (4) loading 100 pL cells / well in KingFisher DW plate. KmgFisher Protocol: (1) attaching the 96 well DW magnet head (use “Change magnet” maintenance protocol); (2) running protocol “conA wash” with Bindlt software; (3) following screen prompts to load tip comb, wash plate, elution plate, and cell plate; (4) pressing “start” to run the program; (5) program specifics: (i) pick-up tip comb, (ii) mix cells, (iii) 1 min 31 sec on slow, (iv) collect beads count 3, 15 sec (v) wash, (vi) Imin 10 sec on medium, (vii) collect beads count 3, 15 sec, (viii) release beads; (6) 3min, fast speed.

[0304] Example 4

[0305] In this example, magnetic beads were bound to cells and were imaged using different imaging software illustrating the affinity of magnetic beads comprising Concanavalin A towards cells. FIG. 14 depicts a plurality of Concanavalin A coated magnetic particles bound to cells and imaged with Countess software, wherein the blue dots are cells, and the black dots are beads. FIGS. 15-20 further illustrate the ability of Concanavalin A magnetic beads to bind to at least one cell using Evos M7000 imaging software. Cytpix imaging software was also used to further demonstrate the ability of Concanavalin A coated magnetic beads to bind to at least one cell as shown in FIGS. 21-26. Therefore, this example illustrates the affinity of Concanavalin A coated magnetic particles towards cells in a sample.

[0306] Example 5

[0307] In this example, Concanavalin A magnetic beads used in cell washing were compared to cell washing performed via centrifugation. “Conical” in FIG. 27 depicts the percent retention of a conical wash starting with 200,000 cells in 1.9 mL; adding 48 mL wash buffer; spinning the 50 mL conical spun at 800 X g for 5 minutes for cells to pellet and removing the liquid, leaving 100 pL; and adding 1 .8 mL wash buffer for final count.

[0308] “Eppendorf ’ in FIG. 27 depicts the percent retention of an Eppendorf wash starting with 200,000 cells in 1.9 mL in a 2 mL Eppendorf tube, wherein the cells were spun down at 800 X g for 5 minutes for the cells to pellet; removing the liquid but leaving 100 pL; adding a 1.9 mL wash to the resuspended cells and spinning the cells down again; and repeating the protocol for a total of three centrifuge spins (final volume was 1.9 mL for final count).

[0309] “Eppendorf ConA” in FIG. 27 depicts the percent retention of a Concanavalin A wash having 200,000 cells bound to 10 pL of Concanavalin A beads (BangsLab) in 1.9 mL; placing the Eppendorf tubes on an Invitrogen™ DynaMag™ 2 magnet for 5 minutes; removing all liquid without touching the visible magnetic beads; removing tubes from magnets and resuspending cells in 2 mL wash buffer and plating back on magnet; and repeating the process for three magnetic binding steps (cells resuspended in 1.9 mL for a final count).

[0310] FIG. 27 establishes that the “Conical” wash had cell retention of from 50% to 65%; the “Eppendorf’ wash had from 65% to 75% cell retention; and the “Eppendorf ConA” wash had a greater than 80% cell retention. Therefore, cells washed using Concanavalin A magnetic particles (no centrifugation) had a much higher rate of cell retention than samples washed via centrifugation.

[0311] Example 6

[0312] In this example, cells were washed via the KingFisher™ Flex Purification System (Thermo Fisher) “KingFisher” and compared to a centrifuge wash (50 mL wash). The “KingFisher” wash had cells bound to Concanavalin A beads (BangsLabs) in a 96 well plate in 100 pL of cell dilution buffer and washed using “Con A wash” protocol on the Bindlt software stored on the King Fisher Flex, which transfers cells and beads into two washes of 250 pL in a 96 well plate and places them into another 96 well plate with 100 pL of cell dilution buffer.

[0313] The centrifuge wash comprised cells starting in a conical; adding 48 mL wash buffer; spinning 50 mL conical at 800 X g for 5 minutes and allowing cells to pellet; removing liquid was removed, leaving 100 pL; and adding 1.8 mL wash buffer for the final count.

[0314] FIG. 28 illustrates cells bound to magnetic particles via Concanavalin A and washed via the KingFisher™ Flex Purification System (Thermo Fisher) had a greater than 70% cell retention, whereas the centrifugation wash via had from 45% cell retention to 60% cell retention.

[0315] Example 7

[0316] In this example, cells, U937 (cell culture suspension cells, human monocytes), PBMC (previously frozen human peripheral blood mononuclear cells in suspension), Hek 293 (cell culture adherent cells, immortalized human embryonic kidney cells), and Hek 293 (cell culture adherent cells, immortalized human embryonic kidney cells) bound to functionalized magnetic particles via a Concanavalin A ligand were washed using the Kingfisher™ Flex Purification System (Thermo Fisher).

[0317] FIG. 29 depicts the cell retention for different cells, with U937 cells having a 75% to a 115% retention; PBM having a 100% to 120% retention; Hek 293 cells having a 65% to 105% retention; and 3T3 having a 95% to 103% retention. Thus, as illustrated by FIG. 26, Concanavalin A beads will bind to many cell types and will stay bound through the cell wash on the KingFisher™ Flex Purification System (Thermo Fisher).

[0318] Example 8

[0319] FIG. 30 is a graph depicting data comparing several aspects illustrating that cells bound to functionalized magnetic particles comprising a Concanavalin A ligand washed at various cell concentrations retained greater than 70% of cells (“Control 100k” and “Control 10k”). The wash titled, “Control 100k,” had 100,000 cells / well in a 96 well dish; and the wash, “Control 10k” had 10,000 cells / well in a 96 well dish. Both samples were washed by extracting the functionalized magnetic particles bound to the cells via Concanavalin A and placing the cells into new containers having a new wash buffer. The data depicted in FIG. 30 illustrates that the cell wash can occur at various cell concentrations with a greater than 70% cell retention when cells are bound to the functionalized magnetic particles via Concanavalin A.

[0320] On the other hand, cells that were washed via centrifugation (not bound to Concanavalin A) had a cell retention of less than 70%, as shown by FIG. 30 (“Centrifuge” illustrating cells washed via centrifugation). The “Centrifuge” wash had 200,000 cells in a conical vial, 48 mL of wash buffer was added, the conical vial was spun down at 800 X g for 5 minutes allowing for the cells to pellet, the liquid was removed (leaving 100 pL), and 1.8 mL of wash buffer was added for the final count. FIG. 30 illustrates that the cell wash with functionalized Concanavalin A coated magnetic particles can occur at various cell concentrations with a greater than 70% retention when cell washing cells proceeds using Concanavalin A coated particles bound to the cells.

[0321] It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

We claim:

1. A method for tagging a nucleic acid within a cell, comprising:(a) contacting a permeabilized cell comprising at least one cellular nucleic acid and a functionalized magnetic particle comprising at least one ligand, to produce a mixture comprising the permeabilized cell associated with the at least one ligand of the functionalized magnetic particle;(b) contacting the at least one cellular nucleic acid with a first oligonucleotide, at least one nucleotide, a first primer, and a first polymerase under conditions to produce a first tagged nucleic acid strand in the permeabilized cell; and(c) applying a magnetic field to the mixture, thereby separating the permeabilized cell associated with the ligand of the functionalized magnetic particle and the first tagged nucleic acid strand from other components of the mixture; wherein steps (a) and (b) can occur in either order.

2. The method of claim 1, wherein the ligand comprises a carbohydrate or a lectin.

3. The method of claim 2, wherein the carbohydrate is selected from the group consisting of dextran, dextran-hydrogel, other dextran derivatives, chitin, chitosan, fochrysolaminarin, xylan, arabinoxylan, mannan, fucoidan, galactomannan, or derivatives thereof or the lectin is selected from the group consisting of abrin, aggrecan, asialoglycoprotein receptor, calnexin, calrecticulin, CD22, CD33, CD94, collectin (mannan-binding lectin), Concanavalin A, gal ectin, Griffonia simplicifolia II agglutinin, legume lectin, mannose receptor, myelin-associated glycoprotein, N-acetylglucosamine receptor, phytohaemagglutinin, Pisum sativum agglutinin, pokeweed mitogen, ricin, selectin, sialoadhesin, soybean agglutinin, Ulex europaeus agglutinin-I, versican, and Vi scum album agglutinin.

4. The method of claim 3, wherein the lectin is Concanavalin A.

5. The method of any one of claims 1 to 4, further comprising contacting the functionalized magnetic particle comprising at least one ligand with an activation buffer to increase affinity of the permeabilized cell to the functionalized magnetic particle.

6. The method of any one of claims 1 to 5, wherein the permeabilized cell is prepared by contacting the cell with a permeabilizing agent to form the permeabilized cell.

7. The method of any one of claims 1 to 6, wherein the permeabilized cell is contacted with a fixation agent or is fixed prior to permeabilization and / or prior to contacting the permeabilized cell with the functionalized magnetic particle comprising the at least one ligand.

8. The method of claim 7, further comprising providing a quenching solution to the fixation agent.

9. The method of any one of claims 1 to 8, wherein the at least one oligonucleotide is at least one oligonucleotide-tethered nucleotide.

10. The method of claim 9, wherein the oligonucleotide-tethered nucleotide comprises dideoxyadenosine triphosphate, dideoxyguanosine triphosphate, dideoxythymidine triphosphate, dideoxyuridine triphosphate, dideoxycytidine triphosphate, or any combination thereof.

11. The method of claim 9 or claim 10, wherein the oligonucleotide-tethered nucleotide comprises Formula 1, or a salt thereofFormula 1, wherein NB is a nucleobase; each of X and Q are independently selected from, H, OH, N3, halo, alkyl, alkoxy, alkyl, alkenyl, alkynyl, acyl, cyano, amino, ester, and amido;Z and Y are independently a bond, amino, amido, alkyl, alkenyl, alkynyl, thioether, sulfonyl, sulfonamido, ether, ketone, carbonyl, anhydride, ester, imido, urea, urethane, and combinations thereof; andCXN is selected from alkylene, alkenylene, alkynylene, ketone, carbonate, ester, ether, anhydride, amido, amino, aminoalkylene, imino, imido, diazo, carbamate ester, phosphodiester, sulfide, disulfide, sulfonyl, sulfonamido, and a heterocyclic group containing from one to four N, O, S atom or a combination thereof.

12. The method of any one of claims 1 to 11, further comprising: contacting the first tagged nucleic acid strand with a second primer, wherein the second primer is partially complementary to the oligonucleotide-tethered nucleotide after producing the first tagged nucleic acid strand, to form an annealed second primer; contacting the first tagged nucleic acid strand and the annealed second primer with a second polymerase and at least one nucleotide not tethered to an oligonucleotide to extend the oligonucleotide-tethered nucleotide sequence, using the second primer as a template to form a second tagged nucleic acid strand, wherein the second tagged nucleic acid strand is present in a mixture comprising the permeabilized cell associated with the ligand of the functionalized magnetic particle; and placing the mixture comprising the permeabilized cell associated with the ligand of the functionalized magnetic particle and the second tagged nucleic acid strand in a magnetic field and separating the permeabilized cell associated with the ligand ofthe functionalized magnetic particle and the second tagged nucleic acid strand from other components of the mixture.

13. The method of any one of claims 1 to 11, further comprising: contacting the first tagged nucleic acid strand with a double-stranded oligonucleotide, wherein one of the nucleotides is partially complementary to the oligonucleotide-tethered nucleotide after producing the first tagged nucleic acid strand, to form an annealed second primer; ligating the non-complementary strand of the double-stranded oligonucleotide to the oligonucleotide-tethered nucleotide, thereby producing a second tagged nucleic acid strand, wherein the second tagged nucleic acid strand is present in a mixture comprising the permeabilized cell associated with the ligand of the functionalized magnetic particle; and placing the mixture comprising the permeabilized cell associated with the ligand of the functionalized magnetic particle and the second tagged nucleic acid strand in a magnetic field, and separating the permeabilized cell associated with the ligand of the functionalized magnetic particle and the second tagged nucleic acid strand from other components of the mixture.

14. The method of any one of claims 1 to 13, wherein the first primer, the second primer, and / or the tethered oligonucleotide comprise a random sequence, a target-specific sequence, or both.

15. The method of any one of claims 1 to 14, wherein the first primer, the second primer, or the tethered oligonucleotide comprise a universal handle, a universal sequence, a unique molecular identifier, an adapter sequence, a promoter sequence, a barcode sequence, an index sequence, or any combination thereof.

16. A method for preparing a nucleic acid library, comprising: preparing a sample comprising a plurality of cells, wherein at least one cell of the plurality of cells comprises a cellular nucleic acid, and wherein the at least one cell is fixed and permeabilized; barcoding the cellular nucleic acid of the at least one fixed and permeabilized cell in the mixture; contacting the sample with a functionalized magnetic particle comprising at least one ligand, producing a mixture wherein the at least one fixed and permeabilized cell is bound to the at least one ligand; applying a magnetic field to the sample, thereby separating the mixture from the other components of the sample; lysing the plurality of cells in the separated mixture; and preparing a nucleic acid library from the lysed cells by providing at least one amplification primer to form an amplified nucleic acid.

17. The method of claim 16, wherein barcoding the cellular nucleic acid comprises: splitting the mixture into a plurality of first containers; preparing nucleic acids that are complementary to the cellular nucleic acid; annealing a first primer to form an annealed first primer which is at least partially complementary to the at least one complementary nucleic acid, the first primer comprising a first universal handle sequence and a first barcode, the first barcode being common to the container, but different from the first barcodes present in the first primers in other containers; and contacting the at least one complementary nucleic acid with a polymerase, at least one nucleotide, and at least one oligonucleotide-tethered dideoxynucleotide, the oligonucleotide of the oligonucleotide-tethered dideoxynucleotide comprising a second universal handle sequence.

18. The method of claim 17, further comprising: forming at least one nucleic acid strand comprising the oligonucleotide-tethered dideoxynucleotide at their 3’ end; pooling the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the least one nucleic acid strands comprising the oligonucleotide-tethered dideoxynucleotide at their 3’ end from the plurality of first containers to provide a first pool; washing the first pool; splitting the first pool into a plurality of second containers; annealing a second primer with the tethered oligonucleotide to form an annealed second primer, wherein the second primer is at least partially complementary to the tethered oligonucleotide, and allowing the polymerase to extend from a 3’ hydroxyl of the annealed second primer to the tethered oligonucleotides to form an extended annealed second primer; pooling the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the extended annealed second primer to provide a second pool; washing the second pool; and splitting the second pool into a plurality of third containers.

19. The method of claim 18, further comprising: forming a first extension product comprising the oligonucleotide-tethered dideoxynucleotide at the 3’ end; pooling the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the formed first extension products comprising the first extension product to form a third pool; washing the third pool; splitting the third pool into a plurality of fourth containers;contacting a splint oligonucleotide with the tethered oligonucleotide of the first extension product, wherein the splint oligonucleotide is partially complementary to the tethered oligonucleotide of the first extension product; contacting the first extension product with a nucleic acid polymerase and one or more nucleotides to allow the polymerase extend across the annealed splint from the 3’ hydroxyl of the tethered oligonucleotide to produce a second extension product; pooling the functionalized magnetic particles bound to the fixed and permeabilized cell comprising the second extension product to form a fourth pool; washing the fourth pool; and splitting fourth pool into a plurality of fifth containers.

20. The method of claim 17, further comprising: forming a first extension product comprising the oligonucleotide-tethered dideoxynucleotide at the 3’ end; pooling the functionalized magnetic particles bound to a permeabilized cell comprising the formed first extension products comprising the first extension product; washing the functionalized magnetic particles bound to a permeabilized cell comprising first extension products comprising the first extension product; splitting the functionalized magnetic particles bound to a permeabilized cell comprising the first extension product into a plurality of second containers; providing a pre-annealed oligonucleotide comprising a second barcode sequence which is at least partially complementary to the tethered oligonucleotide of the first extension products; contacting the first extension products with the pre-annealed oligonucleotide and a ligase to form a ligation product comprising the oligonucleotide-tethered dideoxynucleotide at the 3’ end and a second barcode; pooling the functionalized magnetic particles bound to a permeabilized cell comprising the ligation product; washing the functionalized magnetic particles bound to a permeabilized cell comprising the ligation product; andsplitting the functionalized magnetic particles bound to a permeabilized cell comprising the ligation product into a plurality of third containers.

21. The method of claim 17, wherein the providing at least one amplification primer comprises the at least one amplification primer hybridizing and extending from a first universal handle and a third universal handle22. The method of claim 21, the amplification primers comprising a third barcode, a fourth barcode, a first adaptor sequence, a second adapter sequence, or any combination thereof.

23. The method of claim 22, further comprising generating amplification products, the combination of the first barcode sequence, the second barcode sequence, the third barcode sequence of the amplification products comprising unique sequences to the amplification products originating from a single cell or nucleus.

24. A composition, comprising: a functionalized magnetic particle comprising at least one ligand; at least one cell bound to the at least one ligand, wherein the cell comprises at least one cellular nucleic acid; and at least one nucleic acid complementary to the at least one cellular nucleic acid, wherein the complementary nucleic acid comprises a first barcode and an oligonucleotide tethered nucleotide.

25. The composition of claim 24, wherein the at least one complementary nucleic acid is a cDNA.

26. The compositions of claim 24 or claim 25, wherein the cell is a permeabilized cell.

27. The composition of any one of claims 24 to 26, wherein the at least one functionalized magnetic particle has a diameter of from greater than 0 pm to 100 pm or a diameter from 500 nm to 1500 nm.

28. The composition of any one of claims 24 to 27, wherein the at least one ligand comprises a lectin or a carbohydrate.

29. The composition of claim 28, wherein the carbohydrate is selected from the group consisting of dextran, dextran-hydrogel, other dextran derivatives, chitin, chitosan, fochrysolaminarin, xylan, arabinoxylan, mannan, fucoidan, galactomannan, or derivatives thereof or the lectin is selected from the group consisting of abrin, aggrecan, asialoglycoprotein receptor, calnexin, calrecticulin, CD22, CD33, CD94, collectin (mannan-binding lectin), Concanavalin A, gal ectin, Griffonia simplicifolia II agglutinin, legume lectin, mannose receptor, myelin-associated glycoprotein, N-acetylglucosamine receptor, phytohaemagglutinin, Pisum sativum agglutinin, pokeweed mitogen, ricin, selectin, sialoadhesin, soybean agglutinin, Ulex europaeus agglutinin-I, versican, and Vi scum album agglutinin.

30. The composition of claim 29, wherein the lectin is Concanavalin A.

31. The composition of any one of claims 24 to 30, further comprising at least one enzyme.

32. The composition of claim 31, wherein the at least one enzyme is a polymerase, a ligase, or a reverse transcriptase.

33. The composition of claim 32, wherein the polymerase comprises a DNA polymerase or an RNA polymerase.

34. The composition of any one of claims 24 to 33, wherein the cellular nucleic acid comprises DNA or RNA.

35. The composition of any one of claims 24 to 34, wherein the at least one oligonucleotide tethered nucleotide comprises from 3 nucleotides to 100 nucleotides in length.

36. The composition of any one of claims 24 to 35, wherein the at least one oligonucleotide-tethered nucleotide comprises Formula 1, or a salt thereof:Formula 1, wherein NB is a nucleobase; each of X and Q are independently selected from, H, OH, N3, halo, alkyl, alkoxy, alkyl, alkenyl, alkynyl, acyl, cyano, amino, ester, and amido;Z and Y are independently a bond, amino, amido, alkyl, alkenyl, alkynyl, thioether, sulfonyl, sulfonamido, ether, ketone, carbonyl, anhydride, ester, imido, urea, urethane, and combinations thereof; andCXN is selected from alkylene, alkenylene, alkynylene, ketone, carbonate, ester, ether, anhydride, amido, amino, aminoalkylene, imino, imido, diazo, carbamate ester, phosphodiester, sulfide, disulfide, sulfonyl, sulfonamido, and a heterocyclic group containing from one to four N, O, S atom or a combination thereof.

37. The composition of any one of claims 24 to 36, wherein the oligonucleotide-tethered nucleotide comprises dideoxyadenosine triphosphate, dideoxyguanosine triphosphate, dideoxythymidine triphosphate, dideoxyuridine triphosphate, dideoxycytidine triphosphate, or any combination thereof.

38. A system, comprising: at least one extraction module; at least one container comprising the composition of any one of claims 24 to 37; a positioner to position the extraction module in the container to remove the composition from the container; and a controller having stored instructions for controlling the system.

39. The system of claim 38, wherein the instructions comprise a method for preparing a single cell RNA sequencing library.

40. The system of claim 38 or claim 39, the container comprising a columnar container, a tube, multi-well plate, or any combination thereof.

41. The system of any one of claims 38 to 40, the extraction module comprising at least one magnet.

42. The system of any one of claims 37 to 41, further comprising a magnetic rod configured for operation with a multi-well plate.

43. The system according to claim 42, the wells of the multi -well plate comprising a first end and a second end opposite the first end; the first end comprising an opening configured to the magnetic rod; and the second end comprising a tapered closed end.

44. A kit for preparing a nucleic acid library, the kit comprising: a functionalized magnetic particle comprising at least one ligand; and at least one reaction component for incorporating an oligonucleotide into a nucleic acid of a cell in a sample via a synthesis reaction, wherein the oligonucleotide is an oligonucleotide-tethered nucleotide having Formula 1, or a salt thereof:Formula 1, wherein NB is a nucleobase; each of X and Q are independently chosen from, H, OH, N3, halo, alkyl, alkoxy, alkyl, alkenyl, alkynyl, acyl, cyano, amino, ester, and amido;Z and Y are independently a bond, amino, amido, alkyl, alkenyl, alkynyl, thioether, sulfonyl, sulfonamido, ether, ketone, carbonyl, anhydride, ester, imido, urea, urethane, and combinations thereof; andCXN is chosen from alkylene, alkenylene, alkynylene, ketone, carbonate, ester, ether, anhydride, amido, amino, aminoalkylene, imino, imido, diazo, carbamate ester, phosphodiester, sulfide, disulfide, sulfonyl, sulfonamido, and a heterocyclic group containing from one to four N, O, S atom, or a combination thereof; and instructions for using the functionalized magnetic particles.

45. The kit of claim 44, wherein the ligand comprises a lectin or a carbohydrate.

46. The kit of claim 45, wherein the carbohydrate is selected from the group consisting of dextran, dextran-hydrogel, other dextran derivatives, chitin, chitosan, fochrysolaminarin, xylan, arabinoxylan, mannan, fucoidan, galactomannan, or derivatives thereof or the lectin is selected from the group consisting of abrin, aggrecan, asialoglycoprotein receptor, calnexin, calrecticulin, CD22, CD33, CD94, collectin (mannan-binding lectin), Concanavalin A, gal ectin, Griffonia simplicifolia II agglutinin, legume lectin, mannose receptor, myelin-associated glycoprotein, N-acetylglucosamine receptor, phytohaemagglutinin, Pisum sativum agglutinin, pokeweed mitogen, ricin,selectin, sialoadhesin, soybean agglutinin, Ulex europaeus agglutinin-I, versican, and Vi scum album agglutinin.

47. The kit of any one of claims 44 to 46, further comprising a container comprising the at least one functionalized magnetic particle, at least one oligonucleotide, and the at least one reaction component.

48. The kit of any one of claims 44 to 47, further comprising one or more containers comprising one or more of a binding buffer for activating the at least one functionalized magnetic particle, a buffering agent, a washing buffer, and a lysis buffer.

49. The kit of any one of claims 44 to 48, configured for use with the system of any of claims 38 to 43.

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